U.S. patent application number 13/250141 was filed with the patent office on 2012-01-26 for using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality.
This patent application is currently assigned to University of Connecticut Health Center. Invention is credited to Zihai Li, Pramod K. Srivastava.
Application Number | 20120021996 13/250141 |
Document ID | / |
Family ID | 29272617 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120021996 |
Kind Code |
A1 |
Srivastava; Pramod K. ; et
al. |
January 26, 2012 |
USING HEAT SHOCK PROTEINS TO IMPROVE THE THERAPEUTIC BENEFIT OF A
NON-VACCINE TREATMENT MODALITY
Abstract
The present invention relates to methods of improving a
treatment outcome comprising administering a heat shock protein
(HSP) preparation or an .alpha.-2-macroglobulin (.alpha.2M)
preparation with a non-vaccine treatment modality. In particular,
an HSP preparation or an .alpha.2M preparation is administered in
conjunction with a non-vaccine treatment modality for the treatment
of cancer or infectious diseases. In the practice of the invention,
a preparation comprising HSPs such as but not limited to, hsp70,
hsp90 and gp96 alone or in combination with each other,
noncovalently or covalently bound to antigenic molecules or
.alpha.2M, noncovalently or covalently bound to antigenic molecules
is administered in conjunction with a non-vaccine treatment
modality.
Inventors: |
Srivastava; Pramod K.;
(Avon, CT) ; Li; Zihai; (Avon, CT) |
Assignee: |
University of Connecticut Health
Center
Farmington
CT
|
Family ID: |
29272617 |
Appl. No.: |
13/250141 |
Filed: |
September 30, 2011 |
Related U.S. Patent Documents
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12418724 |
Apr 6, 2009 |
8029808 |
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13250141 |
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11283103 |
Nov 18, 2005 |
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12418724 |
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10322312 |
Dec 16, 2002 |
6984389 |
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10131961 |
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10322312 |
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Current U.S.
Class: |
514/19.3 |
Current CPC
Class: |
A61K 31/506 20130101;
Y02A 50/401 20180101; A61P 35/02 20180101; A61K 31/353 20130101;
A61P 35/00 20180101; A61K 2039/6043 20130101; Y02A 50/407 20180101;
A61K 39/385 20130101; A61K 38/17 20130101; A61K 39/39558 20130101;
A61K 31/196 20130101; Y02A 50/403 20180101; A61K 45/06 20130101;
A61N 5/10 20130101; Y02A 50/466 20180101; Y02A 50/412 20180101;
Y02A 50/30 20180101; A61K 38/1709 20130101; A61K 39/0011 20130101;
A61K 31/277 20130101; Y02A 50/41 20180101; A61P 43/00 20180101;
A61K 31/675 20130101; A61K 31/395 20130101; A61K 31/7048 20130101;
A61K 31/495 20130101; A61K 31/135 20130101; A61K 38/208 20130101;
Y02A 50/386 20180101; A61K 38/1709 20130101; A61K 2300/00 20130101;
A61K 31/506 20130101; A61K 2300/00 20130101; A61K 31/277 20130101;
A61K 2300/00 20130101; A61K 31/675 20130101; A61K 2300/00 20130101;
A61K 38/208 20130101; A61K 2300/00 20130101; A61K 31/395 20130101;
A61K 2300/00 20130101; A61K 31/353 20130101; A61K 2300/00 20130101;
A61K 31/135 20130101; A61K 2300/00 20130101; A61K 31/196 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
514/19.3 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61P 35/00 20060101 A61P035/00 |
Claims
1. A method of treating cancer in a subject comprising: (a)
administering to the subject at least one treatment modality,
wherein said at least one treatment modality comprises a tyrosine
kinase inhibitor; and (b) administering a purified heat shock
protein preparation.
2-33. (canceled)
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 10/131,961, filed Apr. 25, 2002, which is
incorporated by reference herein in its entirety.
1. INTRODUCTION
[0002] The present invention relates to methods of improving a
treatment outcome comprising administering a heat shock protein
(HSP) preparation or an .alpha.-2-macroglobulin (.alpha.2M)
preparation with a non-vaccine treatment modality. In particular,
an HSP preparation or an .alpha.2M preparation is administered in
conjunction with a non-vaccine treatment modality for the treatment
of cancer or infectious diseases. In the practice of the invention,
a preparation comprising HSPs such as but not limited to, hsp70,
hsp90 and gp96 alone or in combination with each other,
noncovalently or covalently bound to antigenic molecules or
.alpha.2M, noncovalently or covalently bound to antigenic molecules
is administered in conjunction with a non-vaccine treatment
modality.
2. BACKGROUND OF THE INVENTION
[0003] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
invention.
2.1. Immune Responses
[0004] An organism's immune system reacts with two types of
responses to pathogens or other harmful agents--humoral response
and cell-mediated response (See Alberts, B. et al., 1994, Molecular
Biology of the Cell. 1195-96). When resting B cells are activated
by antigen to proliferate and mature into antibody-secreting cells,
they produce and secrete antibodies with a unique antigen-binding
site. This antibody-secreting reaction is known as the humoral
response. On the other hand, the diverse responses of T cells are
collectively called cell-mediated immune reactions. There are two
main classes of T cells--cytotoxic T cells and helper T cells.
Cytotoxic T cells directly kill cells that are infected with a
virus or some other intracellular microorganism. Helper T cells, by
contrast, help stimulate the responses of other cells: they help
activate macrophages, dendritic cells and B cells, for example (See
Alberts, B. et al., 1994, Molecular Biology of the Cell. 1228).
Both cytotoxic T cells and helper T cells recognize antigen in the
form of peptide fragments that are generated by the degradation of
foreign protein antigens inside the target cell, and both,
therefore, depend on major histocompatibility complex (MHC)
molecules, which bind these peptide fragments, carry them to the
cell surface, and present them there to the T cells (See Alberts,
B. et al., 1994, Molecular Biology of the Cell. 1228). MHC
molecules are typically found in abundance on antigen-presenting
cells (APCs).
2.3. Chronic Myeloid Leukemia
[0005] Chronic myeloid (myelogenous, myelocytic, granulocytic)
leukemia ("CML") is a cancer of the blood and bone marrow
characterized by overproduction of white blood cells. CML is
characterized by a chronic phase with a median duration of 3 to 5
years when treated with conventional agents and an accelerated or
acute phase of approximately 3 to 6 months duration, inevitably
terminating fatally. Initially, the chronic phase is characterized
by no or few symptoms and signs. However, in the majority of cases,
constitutional symptoms and abnormal physical findings including
extramedullary abnormalities, such as myeloblastomas, eventually
develop.
[0006] CML accounts for 7% to 20% of all leukemias and affects an
estimated 1 to 2/100,000 persons in the general population. The
American Cancer Society estimates that there will be about 4,400
new cases of CML in the United States this year.
[0007] CML is caused by a specific cytogenetic abnormality, the
Philadelphia ("Ph+") chromosome, which results in a clonal
myeloproliferative disorder of pluripotent hematopoietic stem cells
(Faderl et al., 1999, New England 3. Med. 341(3):164-172). The
Ph+chromosome results from a balanced translocation between the
long arms of chromosomes 9 and 22, resulting in the bcr/abl
chimeric gene that expresses an abnormal fusion protein with
altered tyrosine kinase activity.
[0008] Current treatment options for patients in the chronic phase
of CML include busulfan (BUS), hydroxyurea (HU), interferon
(IFN)-based regimens, specific kinase inhibitor for bcr/abl or bone
marrow/stem cell transplantation (BMT) (Silver et al., 1999, Blood
94(5):1517-1536). Until a few years ago, allogeneic BMT was the
treatment of choice for all eligible patients, because it was the
only treatment that appeared to change the natural course of the
disease. Studies showed that at least half of the patients
transplanted remain alive 5 to 10 years after the treatment.
However, this practice was still complicated by the lack of donors,
and the significant transplant related complications such as graft
versus host diseases and infections. IFN-based regimens have also
influenced the natural course of CML. However, IFN-based regiments
alone only offer survival advantage by a median of about 20 months
(Chronic Myeloid Leukemia Trialists' Collaborative Group, 1997, J.
Natl. Cancer Inst. 89(21):1616-20).
[0009] Specific bcr/abl inhibitors such as Gleevec.TM. (imatinib
mesylate, Novartis.TM.) have shown promise in the phase I clinical
trials. Drucker and Lydon, 2000, J. Clin. Invest. 105(1):3-7; Dazzi
et al., 2000, Leukemia 14:419-426; see also Hellman, Principles of
Cancer Management: 6th edition, 2001, DeVita et al., eds., J. B.
Lippencott Company, Philadelphia, which is hereby incorporated by
reference in its entirety, pp. 2443-2444. Gleevec.TM. (imatinib
mesylate) is also known as signal transduction inhibitor 571,
STI-571, and CGP 57148. Based on the phase II studies (Druker et
al., 2001, New England J. Med. 344(14): 1031-1037, and Druker et
al., 2001, New England J. Med. 344(14):1038-1042) the FDA has
approved the use of Gleevec.TM. to treat the following three phases
of CML: chronic phase that is no longer responding to the standard
therapy, interferon; accelerated phase; and myeloid blast crisis.
The long-term efficacy and toxicity, however, remain unknown.
Furthermore, adverse effects have been observed in
Gleevec.TM.-treated patients including edema, hepatotoxicity, and
hematologic toxicity. Physician's Desk Reference (56.sup.th ed.,
2002). In addition, resistance to Gleevec.TM. has already been
described. Le Coutre et al., 2000, Blood 95(5): 1758-1766. Thus,
there is a need in the art for improved methods of treating
CML.
2.4. Heat Shock Proteins
[0010] Heat shock proteins (HSPs), which are also referred to
interchangeably herein as stress proteins, can be selected from
among any cellular protein that satisfies the following criteria.
It is a protein whose intracellular concentration increases when a
cell is exposed to a stressful stimuli, it is capable of binding
other proteins or peptides, it is capable of releasing the bound
proteins or peptides in the presence of adenosine triphosphate
(ATP) or low pH, and it is a protein showing at least 35% homology
with any cellular protein having any of the above properties. HSPs
include constitutively expressed conserved cellular homologs of the
proteins induced by stress. Therefore it is contemplated that
stress proteins/HSPs include other proteins, muteins, analogs, and
variants thereof having at least 35% to 55%, preferably 55% to 75%,
and most preferably 75% to 85% amino acid identity with members of
the three families with the above properties.
[0011] The first stress proteins to be identified were the HSPs. As
their name implies, HSPs are synthesized by a cell in response to
heat shock. To date, three major families of HSPs have been
identified based on molecular weight. The families have been called
hsp60, hsp70 and hsp90 where the numbers reflect the approximate
molecular weight of the stress proteins in kilodaltons. Many
members of these families were found subsequently to be induced in
response to other stressful stimuli including, but not limited to,
nutrient deprivation, metabolic disruption, oxygen radicals, and
infection with intracellular pathogens. (See Welch, May 1993,
Scientific American 56-64; Young, 1990, Annu. Rev. Immunol.
8:401-420; Craig, 1993, Science 260:1902-1903; Gething, et al.,
1992, Nature 355:33-45; and Lindquist, et al., 1988, Annu. Rev.
Genetics 22:631-677), the disclosures of which are incorporated
herein by reference. It is contemplated that lisps/stress proteins
belonging to alt of these three families can be used in the
practice of the instant invention.
[0012] HSPs are intracellular molecules that are abundant, soluble,
and highly conserved. As intracellular chaperones, HSPs participate
in many biochemical pathways of protein maturation and function
active during times of stress and normal cellular homeostasis. Many
stresses can disrupt the three-dimensional structure, or folding,
of a cell's proteins. Left uncorrected, mis-folded proteins form
aggregates that may eventually kill the cell. HSPs bind to those
damaged proteins, helping them refold into their proper
conformations. In normal (unstressed) cellular homeostasis, HSPs
are required for cellular metabolism. HSPs help newly synthesized
polypeptides fold and thus prevent premature interactions with
other proteins. Also, HSPs aid in the transport of proteins
throughout the cell's various compartments.
[0013] The major HSPs can accumulate to very high levels in
stressed cells, but they occur at low to moderate levels in cells
that have not been stressed. For example, the highly inducible
mammalian hsp70 is hardly detectable at normal temperatures but
becomes one of the most actively synthesized proteins in the cell
upon heat shock (Welch et al., 1985, J. Cell. Biol. 101:1198-1211).
In contrast, hsp90 and hsp60 proteins are abundant at normal
temperatures in most, but not all, mammalian cells and are further
induced by heat (Lai at al., 1984, Mol. Cell. Biol. 4:2802-2810;
van Bergen en Henegouwen et al., 1987, Genes Dev. 1:525-531).
[0014] HSPs have been found to have immunological and antigenic
properties. Immunization of mice with gp96 or p84/86 isolated from
a particular tumor rendered the mice immune to that particular
tumor, but not to antigenically distinct tumors (Srivastava, P. K.
et al., 1988, Immunogenetics 28:205-207; Srivastava, P. K. et al.,
1991, Curr. Top. Microbiol. Immunol. 167:109-123). Further, hsp70
was shown to elicit immunity to the tumor from which it was
isolated but not to antigenically distinct tumors. However, hsp70
depleted of peptides was found to lose its specific immunogenic
activity (Udono, M., and Srivastava, P. K., 1993, J. Exp. Med.
178:1391-1396). These observations suggested that the heat shock
proteins are not antigenic per se, but form noncovalent complexes
with antigenic peptides, and the complexes can elicit specific
immunity to the antigenic peptides (Srivastava, P. K., 1993, Adv.
Cancer Res. 62:153-177; Udono, H. et al., 1994, J. Immunol.,
152:5398-5403; Suto, R. et al., 1995, Science, 269:1585-1588).
Recently, hsp60 and hsp70 have been found to stimulate production
of proinflammatory cytokines, such as TNF.alpha. and IL-6, by
monocytes, macrophages, or cytotoxtic T cells (Breloer et al.,
1999, J. Immunol. 162:3141-3147; Chen at, 1999, J. Immunol.
162:3212-3219; Ohashi at, 2000, J. Immunol. 164:558-561; Mea et at,
2000, Nature Medicine, 6:435-442; Todryk et al., 1999, J. Immunol.
163:1398-1408). Hsp70 has also been shown to target immature
dendritic cells and make them more able to capture antigens (Todryk
et at, J. Immunol. 163:1398-1408). It has been postulated that
release of or induction of expression of hsp60 and hsp70, e.g., due
to cell death, may serve to signal that an immune reaction should
be raised (Chen at, 1999, J. Immunol. 162:3212-3219; Ohashi et al.,
2000, J. Immunol. 164:558-561; Todryk et al., 1999, J. Immunol.
163:1398-1408).
[0015] The use of noncovalent complexes of HSP and peptide,
purified from cancer cells, for the treatment and prevention of
cancer has been described in U.S. Pat. Nos. 5,750,119, 5,837,251,
and 6,017,540.
[0016] The use of HSP-peptide complexes for sensitizing antigen
presenting cells in vitro for use in adoptive immunotherapy is
described in U.S. Pat. Nos. 5,985,270 and 5,830,464.
[0017] HSP-peptide complexes can also be isolated from
pathogen-infected cells and used for the treatment and prevention
of infection caused by the pathogen, such as viruses, and other
intracellular pathogens, including bacteria, protozoa, fungi and
parasites; see U.S. Pat. Nos. 5,961,979, and 6,048,530.
[0018] Immunogenic HSP-peptide complexes can also be prepared by in
vitro complexing of HSPs and antigenic peptides, and the uses of
such complexes for the treatment and prevention of cancer and
infectious diseases has been described in U.S. Pat. Nos. 5,935,576,
and 6,030,618. The use of heat shock protein in combination with a
defined antigen for the treatment of cancer and infectious diseases
have also been described in PCT publication WO97/06821 dated Feb.
27, 1997.
[0019] The purification of HSP-peptide complexes from cell lysate
has been described previously; see for example, U.S. Pat. Nos.
5,750,119, and 5,997,873.
2.5. .alpha.2-Macroglobulin
[0020] The .alpha.-macroglobulins are members of a protein
superfamily of structurally related proteins which also comprises
complement components C3, C4 and C5. The human plasma protein
alpha(2)macroglobulin (.alpha.2M) is a 720 kDa homotetrameric
protein primarily known as proteinase inhibitor and plasma and
inflammatory fluid proteinase scavenger molecule (for review see
Chu and Pizzo, 1994, Lab. Invest. 71:792). Alpha (2) macroglobulin
is synthesized as a 1474 amino acid precursor, the first 23 of
which function as a signal sequence that is cleaved to yield a 1451
amino acid mature protein (Kan et al., 1985, Proc. Natl. Acad. Sci.
U.S.A. 82:2282-2286).
[0021] Alpha(2)macroglobulin promiscuously binds to proteins and
peptides with nucleophilic amino acid side chains in a covalent
manner (Chu et al., 1994, Ann. N.Y. Acad. Sci. 737:291-307) and
targets them to cells which express the .alpha.2M receptor
(.alpha.2MR) (Chu and Pizza, 1993, J. Immunol. 150:48). Binding of
.alpha.2M to the .alpha.2MR is mediated by the C-terminal portion
of .alpha.2M (Holtet et al., 1994, FEBS Lett. 344:242-246) and key
residues have been identified (Nielsen et al., 1996, J. Biol. Chem.
271:12909-12912).
[0022] Generally known for inhibiting protease activity, .alpha.2M
binds to a variety of proteases thorough multiple binding sites
(see, e.g., Hall et al., 1981, Biochem. Biophys. Res. Commun.
100(1):8-16). Protease interaction with .alpha.2M results in a
complex structural rearrangement called transformation, which is
the result of a cleavage within the "bait" region of .alpha.2M
after the proteinase becomes "trapped" by thioesters. The
conformational change exposes residues required for receptor
binding, allowing the .alpha.2M-proteinase complex to bind to the
.alpha.2MR. Methylamine can induce similar conformational changes
and cleavage as that induced by proteinases. The uncleaved form of
.alpha.2M, which is not recognized by the receptor, is often
referred to as the "slow" form (s-.alpha.2M). The cleaved form is
referred to as the "fast" form (f-.alpha.2M) (reviewed by Chu et
al., 1994, Ann. N.Y. Acad. Sci. 737:291-307).
[0023] Studies have shown that, in addition to its
proteinase-inhibitory functions, .alpha.2M, when complexed to
antigens, can enhance the antigens' ability to be taken up by
antigen presenting cells such as macrophages and presented to T
cell hybridomas in vitro by up to two orders of magnitude (Chu and
Pizzo, 1994, Lab. Invest. 71:792), and induce T cell proliferation
(Osada et al., 1987, Biochem. Biophys. Res. Commun. 146:26-31).
Further evidence suggests that complexing antigen with .alpha.2M
enhances antibody production by crude spleen cells in vitro (Osada
et al., 1988, Biochem. Biophys. Res. Commun. 150:883) and elicits
an in vivo antibody responses in experimental rabbits (Chu et al.,
1994, J. Immunol. 152:1538-1545) and mice (Mitsuda et al., 1993,
Biochem. Biophys. Res. Commun. 101:1326-1331). However, none of
these studies have shown whether .alpha.2M-antigen complexes are
capable of eliciting cytotoxic T cell responses in vivo.
[0024] .alpha.2M can form complexes with antigens, which are taken
up by antigen presenting cells ("APCs") via the .alpha.2MR, also
known as LDL (low-density lipoprotein) Receptor-Related Protein
("LRP") or CD91 (see PCT/US01/18047, which is incorporated by
reference herein in its entirety). .alpha.2M directly competes for
the binding of heat shock protein gp96 to the .alpha.2MR,
indicating that .alpha.2M and hsps may bind to a common recognition
site on the .alpha.2MR (Binder et al., 2000, Nature Immunology
1(2), 151-154). Additionally, .alpha.2M-antigenic peptide complexes
prepared in vitro can be administered to animals to generate a
cytotoxic T cell response specific to the antigenic molecules
(Binder et al., 2001, J. Immunol. 166:4968-72). Thus, because hsps
and .alpha.2M have a number of common functional attributes, such
as the ability to bind peptide, the recognition and uptake by the
.alpha.2MR, and the stimulation of a cytotoxic T cell response,
.alpha.2M can be used for immunotherapy against cancer and
infectious diseases.
3. SUMMARY OF THE INVENTION
[0025] The present invention is based, in part, on the recognition
that an HSP preparation can enhance or improve the therapeutic
benefit of non-vaccine treatment modalities or therapeutic
modalities for treatment of cancer or infectious diseases. Thus,
the present invention encompasses methods and compositions that
comprise administering an HSP preparation in combination with a
non-vaccine treatment modality. Also encompassed are methods and
compositions that comprise administering an .alpha.2M preparation
in combination with a non-vaccine treatment modality. In
particular, the invention encompasses methods and compositions of
treatment and compositions that provide a better therapeutic
profile than that of an HSP preparation or .alpha.2M preparation
administered alone or a non-vaccine treatment modality administered
alone. The source of the HSP or .alpha.2M is preferably an
eukaryote, and most preferably a mammal. The subject receiving the
treatment is preferably a mammal including, but not limited to,
domestic animals, such as cats and dogs; wild animals, including
foxes and raccoons; livestock and fowl, including horses, cattle,
sheep, turkeys and chickens, as well as any rodents. Most
preferably, the subject is human.
[0026] The invention provides methods for improving the therapeutic
outcome of a non-vaccine treatment modality comprising
administering either an HSP preparation or an .alpha.2M
preparation, preferably a purified HSP preparation or a purified
.alpha.2M preparation, in conjunction with the administration of
the treatment modality. Either the HSP preparation or the .alpha.2M
preparation can be administered over a period of time which may
precede, overlap, and/or follow a treatment regimen with a
non-vaccine treatment modality. The HSP preparation or the
.alpha.2M preparation can be administered concurrently, before, or
after the administration of the treatment modality. Examples of
treatment modalities include but are not limited to antibiotics,
antivirals, antifungal compounds, anti-cancer treatments such as
chemotherapeutic agents, and radiation, as well as biological
therapeutic agents and immunotherapeutic agents. In preferred
embodiments, the treatment modality is useful in the treatment or
prevention of cancer. In particularly preferred embodiments, the
treatment modality is useful in the treatment or prevention of
chronic myelgenous leukemia or soft tissue sarcomas including but
not limited to gastrointestinal stromal tumors. In another
preferred embodiment, the treatment modality is Gleevec.TM..
[0027] In one embodiment, the invention encompasses methods of
treatment that provide better therapeutic profiles than the
administration of the treatment modality or the HSP preparation
alone. In another embodiment, the invention encompasses methods of
treatment that provide better therapeutic profiles than the
administration of the treatment modality or the .alpha.2M
preparation alone. Encompassed by the invention are methods wherein
the administration of a treatment modality with an HSP preparation
or an .alpha.2M preparation has additive potency or additive
therapeutic effect. The invention also encompasses synergistic
outcomes where the therapeutic efficacy is greater than additive.
Preferably, such administration of a treatment modality with an HSP
preparation or with an .alpha.2M preparation also reduces or avoids
unwanted or adverse effects. Given the invention, in certain
embodiments, doses of non-vaccine treatment modality can be reduced
or administered less frequently, preferably increasing patient
compliance, improving therapy and/or reducing unwanted or adverse
effects. In a specific embodiment, lower or less frequent doses of
chemotherapy or radiation therapy are administered to reduce or
avoid unwanted effects. Alternatively, doses of HSP preparation and
doses of .alpha.2M preparation can be reduced or administered less
frequently if administered with a treatment modality.
[0028] In one embodiment, the present invention provides a method
for improving the outcome of a treatment in a subject receiving a
therapeutic modality which is not a vaccine. The method comprises
administering either a heat shock protein preparation, preferably a
purified HSP preparation, or an .alpha.2M preparation, preferably a
purified .alpha.2M preparation, to the subject before, concurrently
with, or after the administration of the therapeutic modality. In a
specific embodiment, the HSP preparation or the .alpha.2M
preparation can augment the therapeutic benefit of a treatment
modality and improve the outcome of the treatment. Without being
bound by any theory or mechanism, the administration of a mammalian
HSP preparation or .alpha.2M preparation to a subject can enhance
the responsiveness of non-specific immune mechanisms of the
subject, for example, by increasing the number of natural killer
(NK) cells and/or accelerating the maturation of dendritic cells
and/or can also enhance the responsiveness of specific immune
mechanisms, such as by increasing the number of CD4+ and CD8+ T
cells. In a preferred specific embodiment, the HSP preparation is
administered before the administration of the therapeutic modality.
In another preferred specific embodiment, the .alpha.2M preparation
is administered before the administration of the therapeutic
modality.
[0029] In another embodiment, the present invention provides a
method for improving the outcome of a treatment in a subject
receiving an HSP preparation, preferably a purified HSP
preparation, by administering a non-vaccine therapeutic modality to
the subject before, concurrently with, or after the administration
of the HSP preparation. In a specific embodiment, the non-vaccine
therapeutic modality can augment the therapeutic benefit of an HSP
preparation and improve the outcome of the treatment.
[0030] In another embodiment, the present invention provides a
method for improving the outcome of a treatment in a subject
receiving an .alpha.2M preparation, preferably a purified .alpha.2M
preparation, by administering a non-vaccine therapeutic modality to
the subject before, concurrently with, or after the administration
of the .alpha.2M preparation. In a specific embodiment, the
non-vaccine therapeutic modality can augment the therapeutic
benefit of an .alpha.2M preparation and improve the outcome of the
treatment.
[0031] In certain embodiments, the administration of the
HSP/.alpha.2M preparation in the absence of administration of the
therapeutic modality or the administration of the therapeutic
modality in the absence of administration of the HSP/.alpha.2M
preparation is not therapeutically effective. In a specific
embodiment, the amount of HSP/.alpha.2M preparation or therapeutic
modality is administered in an amount insufficient to be
therapeutically effective alone. In alternate embodiments, both or
at least one of the HSP/.alpha.2M preparation or therapeutic
modality is therapeutically effective when administered alone.
[0032] In various embodiments, the methods comprise the
administration of an HSP preparation, preferably a purified HSP
preparation, to a subject receiving a treatment modality for the
treatment of cancer or infectious diseases. Preferably the HSP
preparation comprises HSP-peptide complexes displaying the
antigenicity of a tumor specific antigen or tumor associated
antigen of the type of cancer or an antigen of an infectious agent,
i.e., heat shock proteins complexed to antigenic peptides of the
cancer cells or infected cells from which the complexes are
obtained. Accordingly, in one embodiment, the specific
immunogenicity of the HSP preparation derives from the peptide
complexed to the HSP. In preferred embodiments, the HSP-peptide
complexes are isolated from an antigen source such as cancer
tissues, cancer cells, or infected tissues. In the practice of the
invention, such HSP-peptide complexes are preferably, autologous to
the individual subject, i.e., obtained from the tissues of the
subject receiving the administration of HSP preparation and
treatment modality, but need not be (i.e., allogeneic to the
individual subject).
[0033] In various other embodiments, the methods comprise the
administration of an .alpha.2M preparation, preferably a purified
.alpha.2M preparation, to a subject receiving a treatment modality
for the treatment of cancer or infectious diseases. Preferably the
.alpha.2M preparation comprises .alpha.2M-peptide complexes
displaying the antigenicity of a tumor specific antigen or tumor
associated antigen of the type of cancer or an antigen of an
infectious agent, i.e., .alpha.2M complexed to antigenic peptides
of the cancer cells or infected cells from which the complexes are
obtained. Accordingly, in one embodiment, the specific
immunogenicity of the .alpha.2M preparation derives from the
peptide complexed to the .alpha.2M. In preferred embodiments, the
.alpha.2M-peptide complexes are isolated from an antigen source
such as cancer tissues, cancer cells, or infected tissues. In the
practice of the invention, such .alpha.2M-peptide complexes are
preferably, autologous to the individual subject, i.e., obtained
from the tissues of the subject receiving the administration of
.alpha.2M preparation and treatment modality, but need not be
(i.e., allogeneic to the individual subject).
[0034] In one embodiment, the methods comprise the administration
of an HSP preparation or an .alpha.2M preparation, preferably a
purified HSP preparation or a purified .alpha.2M preparation, to a
subject receiving a treatment modality for treatment of an
infectious disease. Such treatment modalities are known in the art
and include but are not limited to antibiotics, antivirals,
antifungals as well as biological and immunotherapeutic agents.
Preferably the HSP preparation comprises HSP-peptide complexes
which display the antigenicity of an agent of the infectious
disease. Preferably the .alpha.2M preparation comprises
.alpha.2M-peptide complexes which display the antigenicity of an
agent of the infectious disease. In a specific embodiment, the
outcome of a treatment of a type of infectious disease in a subject
receiving a non-vaccine therapeutic modality is improved by
administering HSP-peptide complexes comprising an HSP complexed to
a peptide that displays the antigenicity of an antigen of an agent
of said type of infectious disease. Preferably, the HSP-peptide
complexes are not present in admixture with HSP or .alpha.2M that
is not complexed to a peptide that displays the antigenicity of an
antigen of an agent of the same infectious disease. (See
International Application No. PCT/US01/28840, filed Sep. 15, 2001,
incorporated by reference herein in its entirety). In one
embodiment, the HSP preparation is administered prior to
administration of the therapeutic modality. In another embodiment,
the therapeutic modality is administered prior to the
administration of the HSP preparation. In another specific
embodiment, the outcome of a treatment of a type of infectious
disease in a subject receiving a non-vaccine therapeutic modality
is improved by administering .alpha.2M-peptide complexes comprising
an .alpha.2M complexed to a peptide that displays the antigenicity
of an antigen of an agent of said type of infectious disease.
Preferably, the .alpha.2M-peptide complexes are not present in
admixture with HSP or .alpha.2M that is not complexed to a peptide
that displays the antigenicity of an antigen of an agent of the
same infectious disease. In one embodiment, the .alpha.2M
preparation is administered prior to administration of the
therapeutic modality. In another embodiment the therapeutic
modality is administered prior to the administration of the
.alpha.2M preparation.
[0035] In another embodiment, the methods comprise the
administration of either an HSP preparation or an .alpha.2M
preparation, preferably a purified HSP preparation or a purified
.alpha.2M preparation, to a subject receiving a treatment modality
for treatment of cancer. Such treatment modalities include but are
not limited to chemotherapies and radiation therapies as well as
hormonal therapies, biological therapies and immunotherapies.
Preferably the HSP preparation or .alpha.2M preparation is
administered to a subject receiving chemotherapy or radiation
therapy for treatment of cancer. Preferably the HSP preparation
comprises HSP-peptide complexes which display the antigenicity of
the type of cancer being treated. Preferably where the preparation
is an .alpha.2M preparation, the .alpha.2M preparation comprises
.alpha.2M-peptide complexes which display the antigenicity of the
type of cancer being treated. Accordingly, in preferred
embodiments, the invention provides methods for improving the
outcome of cancer treatment in a subject receiving a therapeutic
modality which is not a vaccine using HSP-peptide complexes
comprising an HSP complexed to a peptide that displays the
antigenicity of a tumor specific antigen or tumor associated
antigen of a type of cancer or using .alpha.2M-peptide complexes
comprising an .alpha.2M complexed to a peptide that displays the
antigenicity of a tumor specific antigen or tumor associated
antigen of a type of cancer. In certain preferred embodiments, such
HSP-peptide complexes and .alpha.2M-peptide complexes are not
diluted with either HSP or .alpha.2M that is not complexed to a
peptide that displays the antigenicity of an antigen of the same
type of cancer. In one embodiment, the HSP preparation or .alpha.2M
preparation is administered prior to administration of the
therapeutic modality. In another embodiment, the therapeutic
modality is administered prior to administration of the HSP
preparation or .alpha.2M preparation.
[0036] In various embodiments, the HSP preparation or .alpha.2M
preparation is administered with an anti-cancer agent which can be
but is not limited to a cytotoxic agent, antimitotic agent, tubulin
stabilizing agent, microtubule formation inhibiting agent,
topoisomerase inhibitors, alkylating agent, DNA interactive agent,
antimetabolite, RNA/DNA antimetabolite, DNA antimetabolite. In a
specific embodiment, the anti-cancer agent is a
chemotherapeutic.
[0037] In a specific embodiment, an HSP preparation is administered
to a subject receiving a chemotherapeutic agent for treatment of
cancer. In another preferred embodiment, an .alpha.2M preparation
is administered to a subject receiving a chemotherapeutic agent for
treatment of cancer. Such chemotherapeutic agents are known in the
art and include but are not limited to: methotrexate, taxol,
mercaptopurine, thioguanine, hydroxyurea, cytarabine,
cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,
mitomycin, dacarbazine, procarbizine, etoposides, campathecins,
bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin,
plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine,
vinorelbine, paclitaxel, and docetaxel, doxorubicin, epirubicin,
5-fluorouracil, taxanes such as docetaxel and paclitaxel,
leucovorin, levamisole, irinotecan, estramustine, etoposide,
nitrosoureas such as carmustine and lomustine, vinca alkaloids,
platinum compounds, mitomycin, gemcitabine, hexamethylmelamine,
topotecan, tyrosine kinase inhibitors, tyrphostins, STI-571 or
Gleevec.TM. (imatinib mesylate), herbimycin A, genistein,
erbstatin, and lavendustin A.
[0038] In preferred embodiments, each of the methods above comprise
administering either an HSP preparation or an .alpha.2M
preparation, preferably a purified HSP preparation or a purified
.alpha.2M preparation, to a subject receiving a drug of the
2-phenylaminopyrimidine class for treatment of cancer. More
preferably, the subject is receiving Gleevec.TM. (i.e., imatinib
mesylate) for treatment of cancer.
[0039] In another specific embodiment, an HSP preparation or an
.alpha.2M preparation is administered to a subject receiving
radiation therapy for treatment of cancer. For radiation treatment,
the radiation can be gamma rays or X-rays. The methods encompass
treatment of cancer comprising radiation therapy, such as
external-beam radiation therapy, interstitial implantation of
radioisotopes (I-125, palladium, iridium), radioisotopes such as
strontium-89, thoracic radiation therapy, intraperitoneal P-32
radiation therapy, and/or total abdominal and pelvic radiation
therapy. For a general overview of radiation therapy, see Hellman,
Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th
edition, 2001, DeVita et al., eds., J.B. Lippencott Company,
Philadelphia. In preferred embodiments, the radiation treatment is
administered as external beam radiation or teletherapy wherein the
radiation is directed from a remote source. In various preferred
embodiments, the radiation treatment is administered as internal
therapy or brachytherapy wherein a radioactive source is placed
inside the body close to cancer cells or a tumor mass.
[0040] In another embodiment, each of the above methods comprise
the administration of HSP preparation, preferably a purified HSP
preparation, to a subject receiving a combination of treatment
modalities for the treatment of cancer. In another embodiment, each
of the above methods comprise the administration of an .alpha.2M
preparation, preferably a purified .alpha.2M preparation, to a
subject receiving a combination of treatment modalities for the
treatment of cancer. Preferably the HSP preparation and .alpha.2M
preparation each comprises HSP-peptide complexes and
.alpha.2M-peptide complexes, respectively, which display the
antigenicity of the type of cancer being treated. In one such
embodiment, an HSP preparation is administered to a subject
receiving chemotherapy in combination with a biological therapy,
preferably a cytokine. In another such embodiment, an .alpha.2M
preparation is administered to a subject receiving chemotherapy in
combination with a biological therapy, preferably a cytokine. In
various embodiments, the cytokine is selected from the group
consisting of IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IFN.alpha., IFN.beta.,
IFN.gamma., TNF.alpha., TNF.beta., G-CSF, GM-CSF, TGF-.beta.,
IL-15, IL-18, GM-CSF, INF-.gamma., INF-.alpha., SLC, endothelial
monocyte activating protein-2 (EMAP2), MIP-3.alpha., MIP-3.beta.,
or an MHC gene, such as HLA-B7. Additionally, other exemplary
cytokines include other members of the TNF family, including but
not limited to TNF-.alpha.-related apoptosis-inducing ligand
(TRAIL), TNF-.alpha.-related activation-induced cytokine (TRANCE),
TNF-.alpha.-related weak inducer of apoptosis (TWEAK), CD40 ligand
(CD40L), LT-.alpha., LT-.beta., OX4OL, CD4OL, FasL, CD27L, CD30L,
4-1BBL, APRIL, LIGHT, TL1, TNFSF16, TNFSF17, and AITR-L, or a
functional portion thereof. See, e.g., Kwon et at., 1999, Curr.
Opin. Immunol. 11:340-345 for a general review of the TNF family.
In one embodiment, the HSP preparation is administered prior to the
treatment modalities. In another embodiment, the treatment modality
is administered prior to the HSP preparation.
[0041] In a preferred embodiment, a purified HSP preparation is
administered to a subject receiving cyclophosphamide in combination
with IL-12 for treatment of cancer. In another preferred
embodiment, a purified .alpha.2M preparation is administered to a
subject receiving cyclophosphamide in combination with IL-12 for
treatment of cancer.
[0042] In another embodiment, the above methods are useful for the
prevention of cancer or infectious diseases. In a specific
embodiment, an HSP preparation is administered in conjunction with
a non-vaccine treatment modality to a subject to reduce the risk of
acquiring a type of cancer or an infectious disease. In other
specific embodiments, the methods encompass administration of an
HSP preparation with administration of a non-vaccine treatment
modality as a preventative measure to a subject having a genetic or
non-genetic predisposition to a cancer or infectious disease or to
a subject facing exposure to an agent of an infectious disease. In
further embodiments, the invention also provides that each of the
foregoing embodiments also can be applicable wherein an .alpha.2M
preparation is administered in conjunction with a non-vaccine
treatment modality.
[0043] The methods and compositions of the invention are useful not
only in untreated patients, but are also useful in the treatment of
patients partially or completely un-responsive to the therapeutic
modality in the absence of the HSP/.alpha.2M preparation or to the
HSP/.alpha.2M preparation in the absence of the therapeutic
modality. In various embodiments, the invention provides methods
and compositions useful in the treatment or prevention of diseases
and disorders in patients that have been shown to be or may be
refractory or non-responsive to therapies comprising the
administration of either or both the HSP/.alpha.2M preparation or
the therapeutic modality. The invention also includes methods and
compositions comprising administration of the HSP/.alpha.2M
preparation and the therapeutic modality to patients that have
previously received and/or are concurrently receiving other forms
of medical therapy.
[0044] The HSP preparation used in the methods and compositions of
the invention is preferably purified, and can include free HSP not
bound to any molecule, and molecular complexes of HSP with another
molecule, such as a peptide. An HSP-peptide complex comprises an
HSP covalently or noncovalently attached to a peptide. The methods
of the invention may or may not require covalent or noncovalent
attachment of an HSP to any specific antigens or antigenic peptides
prior to administration to a subject. Although, the peptide(s) may
be unrelated to the infectious disease or disorder or particular
cancer being treated, in preferred embodiments, the HSP preparation
comprises complexes which display the antigenicity of an antigen of
the agent of infectious disease or of a tumor specific antigen or
tumor associated antigen of the type of cancer being treated,
respectively. More preferably, for the treatment of infectious
disease, the HSP preparation comprises noncovalent HSP-peptide
complexes isolated from a cell infected with an infectious agent
(or non-infectious variant thereof displaying the antigenicity
thereof) that causes the infectious disease. More preferably, for
treatment of a type of cancer, the HSP preparation comprises
noncovalent HSP-peptide complexes isolated from cancerous tissue of
said type of cancer or a metastasis thereof, which can be from the
patient (autologous) or not (allogeneic). Accordingly, for the
purposes of this invention, an HSP preparation is a composition
comprising HSPs whether unbound or bound to other molecules (e.g.,
peptides). The HSP is preferably purified. An HSP preparation may
include crude cell lysate comprising HSP, the amount of lysate
corresponding to between 100 to 10.sup.8 cell equivalents. HSPs can
be conveniently purified from most cellular sources as a population
of complexes of different peptides non-covalently bound to HSPs.
The HSPs can be separated from the non-covalently bound peptides by
exposure to low pH and/or adenosine triphosphate, or other methods
known in the art.
[0045] The .alpha.2M preparation used in the methods and
compositions of the invention is preferably purified, and can
include free .alpha.2M not bound to any molecule, and molecular
complexes of .alpha.2M with another molecule, such as a peptide. An
.alpha.2M-peptide complex comprises an .alpha.2M covalently or
noncovalently attached to a peptide. The methods of the invention
may or may not require covalent or noncovalent attachment of an
.alpha.2M to any specific antigens or antigenic peptides prior to
administration to a subject. Although, the peptide(s) may be
unrelated to the infectious disease or disorder or particular
cancer being treated, in preferred embodiments, the .alpha.2M
preparation comprises complexes which display the antigenicity of
an antigen of the agent of infectious disease or of a tumor
specific antigen or tumor associated antigen of the type of cancer
being treated, respectively. More preferably, for the treatment of
infectious disease, the .alpha.2M preparation comprises noncovalent
.alpha.2M-peptide complexes isolated from a cell infected with an
infectious agent (or non-infectious variant thereof displaying the
antigenicity thereof) that causes the infectious disease. More
preferably, for treatment of a type of cancer, the .alpha.2M
preparation comprises noncovalent .alpha.2M-peptide complexes
isolated from cancerous tissue of said type of cancer or a
metastasis thereof, which can be from the patient (autologous) or
not (allogeneic). Accordingly, for the purposes of this invention,
an .alpha.2M preparation is a composition comprising .alpha.2M
whether unbound or hound to other molecules (e.g., peptides). The
.alpha.2M is preferably purified. An .alpha.2M preparation may
include crude cell lysate comprising .alpha.2M, the amount of
lysate corresponding to between 100 to 10.sup.8 cell equivalents.
.alpha.2M s can be conveniently purified from most cellular sources
as a population of complexes of different peptides non-covalently
bound to .alpha.2Ms. The .alpha.2M can be separated from the
non-covalently bound peptides by exposure to low pH and/or
adenosine triphosphate, or other methods known in the art.
[0046] In various embodiments, the source of the HSP and the
.alpha.2M is preferably an eukaryote, more preferably a mammal, and
most preferably a human. Accordingly, the HSP preparation used by
the methods of the invention includes eukaryotic HSPs, mammalian
HSPs and human HSPs. The .alpha.2M preparation includes eukaryotic
.alpha.2M, mammalian .alpha.2M and human .alpha.2M. The eukaryotic
source from which the HSP preparation or .alpha.2M preparation is
derived and the subject receiving the HSP preparation or the
.alpha.2M preparation, respectively, are preferably the same
species.
[0047] In one embodiment, the specific immunogenicity of the HSP
preparation derives from the peptide complexed to a heat shock
protein. Accordingly, in various embodiments, the HSP preparation
comprises heat shock protein peptide complexes wherein the heat
shock proteins are complexed to peptides derived from a specific
antigen source. In a preferred embodiment, the HSP protein
preparation comprises heat shock protein-peptide complexes that are
autologous. In another preferred embodiment, the HSP preparation
comprises heat shock proteins complexed to antigenic peptides of
the cancer cells from which they are derived. In specific
embodiments, the antigen is a tumor specific antigen (i.e., only
expressed in the tumor cells). In other specific embodiments, the
antigen is a tumor associated antigen (i.e., relatively
overexpressed in the tumor cells). In yet another preferred
embodiment, the HSP preparation comprises heat shock proteins
complexed to antigenic peptides of the infected cells from which
they are derived.
[0048] In another embodiment, the specific immunogenicity of the
.alpha.2M preparation derives from the peptide complexed to an
.alpha.2M. Accordingly, in various embodiments, the .alpha.2M
preparation comprises .alpha.2M peptide complexes wherein the
.alpha.2M are complexed to peptides derived from a specific antigen
source. In a preferred embodiment, the .alpha.2M protein
preparation comprises .alpha.2M-peptide complexes that are
autologous. In another preferred embodiment, the .alpha.2M
preparation comprises .alpha.2M complexed to antigenic peptides of
the cancer cells from which they are derived. In other specific
embodiments, the antigen is a tumor associated antigen (i.e.,
relatively overexpressed in the tumor cells). In yet another
preferred embodiment, the .alpha.2M preparation comprises .alpha.2M
complexed to antigenic peptides of the infected cells from which
they are derived.
[0049] Also encompassed by the invention are methods of treatment
and delivery, pharmaceutical compositions and formulas comprising
administering at least one non-vaccine therapeutic modality and an
HSP preparation or an .alpha.2M preparation and kits comprising
such pharmaceutical compositions.
4. DESCRIPTION OF THE FIGURE
[0050] FIG. 1. Synopsis of clinical protocol described in section
7, infra. The synopsis includes all physical examinations, blood
work, x-rays and bone marrow tests that were done before, during
and after HSP-peptide complex vaccination.
5. DETAILED DESCRIPTION OF THE INVENTION
[0051] The present invention is based, in part, on the recognition
that an HSP preparation can enhance or improve the therapeutic
benefit of non-vaccine treatment modalities or therapeutic
modalities for treatment of cancer or infectious diseases. Thus,
the present invention encompasses methods and compositions that
comprise administering an HSP preparation in combination with a
non-vaccine treatment modality. Also encompassed are methods and
compositions that comprise administering an .alpha.2M preparation
in combination with a non-vaccine treatment modality. In
particular, the invention encompasses methods of treatment and
compositions that provide a better therapeutic profile than that of
an HSP preparation or .alpha.2M preparation administered alone or a
non-vaccine treatment modality administered alone. The source of
the HSP or .alpha.2M is preferably an eukaryote, and most
preferably a mammal. The subject receiving the treatment is
preferably a mammal including, but not limited to, domestic
animals, such as cats and dogs; wild animals, including foxes and
raccoons; livestock and fowl, including horses, cattle, sheep,
turkeys and chickens, as well as any rodents. Most preferably, the
subject is human.
[0052] The invention provides methods for improving the therapeutic
outcome of a non-vaccine treatment modality comprising
administering either an HSP preparation or an .alpha.2M
preparation, preferably a purified HSP preparation or a purified
.alpha.2M preparation, in conjunction with the administration of
the treatment modality. Either the HSP preparation or the .alpha.2M
preparation can be administered over a period of time which may
precede, overlap, and/or follow a treatment regimen with a
non-vaccine treatment modality. The HSP preparation or the
.alpha.2M preparation can be administered concurrently, before, or
after the administration of the treatment modality. Examples of
treatment modalities include but are not limited to antibiotics,
antivirals, antifungal compounds, anti-cancer treatments such as
chemotherapeutic agents, and radiation, as well as biological
therapeutic agents and immunotherapeutic agents. In preferred
embodiments, the treatment modality is useful in the treatment or
prevention of cancer. In another preferred embodiment, the
treatment modality is Gleevec.TM..
[0053] In one embodiment, the invention encompasses methods of
treatment that provide better therapeutic profiles than the
administration of the treatment modality or the HSP preparation
alone. In another embodiment, the invention encompasses methods of
treatment that provide better therapeutic profiles than the
administration of the treatment modality or the .alpha.2M
preparation alone. Encompassed by the invention are methods wherein
the administration of a treatment modality with an HSP preparation
or an .alpha.2M preparation has additive potency or additive
therapeutic effect. The invention also encompasses synergistic
outcomes where the therapeutic efficacy is greater than additive.
Preferably, such administration of a treatment modality with an HSP
preparation or with an .alpha.2M preparation also reduces or avoids
unwanted or adverse effects. Given the invention, in certain
embodiments, doses of non-vaccine treatment modality can be reduced
or administered less frequently, preferably increasing patient
compliance, improving therapy and/or reducing unwanted or adverse
effects. In a specific embodiment, lower or less frequent doses of
chemotherapy or radiation therapy are administered to reduce or
avoid unwanted effects. Alternatively, doses of HSP preparation and
doses of .alpha.2M preparation can be reduced or administered less
frequently if administered with a treatment modality.
[0054] In one embodiment, the present invention provides a method
for improving the outcome of a treatment in a subject receiving a
therapeutic modality which is not a vaccine. The method comprises
administering either a heat shock protein preparation, preferably a
purified HSP preparation, or an .alpha.2M preparation, preferably a
purified .alpha.2M preparation, to the subject before, concurrently
with, or after the administration of the therapeutic modality. In a
specific embodiment, the HSP preparation or the .alpha.2M
preparation can augment the therapeutic benefit of a treatment
modality and improve the outcome of the treatment. Without being
bound by any theory or mechanism, the administration of a mammalian
HSP preparation or .alpha.2M preparation to a subject can enhance
the responsiveness of non-specific immune mechanisms of the
subject, for example, by increasing the number of natural killer
(NK) cells and/or accelerating the maturation of dendritic cells
and/or can also enhance the responsiveness of specific immune
mechanisms, such as by increasing the number of CD4+ and CD8+ T
cells. In a specific embodiment, the HSP preparation is
administered before the administration of the therapeutic modality.
In another specific embodiment, the therapeutic modality is
administered before the administration of the HSP preparation. In
specific embodiment, the .alpha.2M preparation is administered
before the administration of the therapeutic modality. In another
specific embodiment, the therapeutic modality is administered
before the administration of the .alpha.2M preparation.
[0055] In another embodiment, the present invention provides a
method for improving the outcome of a treatment in a subject
receiving an HSP preparation, preferably a purified HSP
preparation, by administering a non-vaccine therapeutic modality to
the subject before, concurrently with, or after the administration
of the HSP preparation. In a specific embodiment, the non-vaccine
therapeutic modality can augment the therapeutic benefit of an HSP
preparation and improve the outcome of the treatment.
[0056] In another embodiment, the present invention provides a
method for improving the outcome of a treatment in a subject
receiving an .alpha.2M preparation, preferably a purified .alpha.2M
preparation, by administering a non-vaccine therapeutic modality to
the subject before, concurrently with, or after the administration
of the .alpha.2M preparation. In a specific embodiment, the
non-vaccine therapeutic modality can augment the therapeutic
benefit of an .alpha.2M preparation and improve the outcome of the
treatment.
[0057] In certain embodiments, the administration of the
HSP/.alpha.2M preparation in the absence of administration of the
therapeutic modality or the administration of the therapeutic
modality in the absence of administration of the HSP/.alpha.2M
preparation is not therapeutically effective. In a specific
embodiment, the amount of HSP/.alpha.2M preparation or therapeutic
modality is administered in an amount insufficient to be
therapeutically effective alone. In alternate embodiments, both or
at least one of the HSP/.alpha.2M preparation or therapeutic
modality is therapeutically effective when administered alone.
[0058] In various embodiments, the methods comprise the
administration of an HSP preparation, preferably a purified HSP
preparation, to a subject receiving a treatment modality for the
treatment of cancer or infectious diseases. Preferably the HSP
preparation comprises HSP-peptide complexes displaying the
antigenicity of a tumor specific antigen or tumor associated
antigen of the type of cancer or an antigen of an infectious agent,
i.e., heat shock proteins complexed to antigenic peptides of the
cancer cells or infected cells from which the complexes are
obtained. Accordingly, in one embodiment, the specific
immunogenicity of the HSP preparation derives from the peptide
complexed to the HSP. In preferred embodiments, the HSP-peptide
complexes are isolated from an antigen source such as cancer
tissues or infected tissues. In the practice of the invention, such
HSP-peptide complexes are preferably, autologous to the individual
subject, i.e., obtained from the tissues of the subject receiving
the administration of HSP preparation and treatment modality, but
need not be (i.e., allogeneic to the individual subject).
[0059] In various other embodiments, the methods comprise the
administration of an .alpha.2M preparation, preferably a purified
.alpha.2M preparation, to a subject receiving a treatment modality
for the treatment of cancer or infectious diseases. Preferably the
.alpha.2M preparation comprises .alpha.2M-peptide complexes
displaying the antigenicity of a tumor specific antigen or tumor
associated antigen of the type of cancer or an antigen of an
infectious agent, i.e., .alpha.2M complexed to antigenic peptides
of the cancer cells or infected cells from which the complexes are
obtained. Accordingly, in one embodiment, the specific
immunogenicity of the .alpha.2M preparation derives from the
peptide complexed to the .alpha.2M. In preferred embodiments, the
.alpha.2M-peptide complexes are isolated from an antigen source
such as cancer tissues or infected tissues. In the practice of the
invention, such .alpha.2M-peptide complexes are preferably,
autologous to the individual subject, i.e., obtained from the
tissues of the subject receiving the administration of .alpha.2M
preparation and treatment modality, but need not be (i.e.,
allogeneic to the individual subject).
[0060] In one embodiment, the methods comprise the administration
of an HSP preparation or an .alpha.2M preparation, preferably a
purified HSP preparation or a purified .alpha.2M preparation, to a
subject receiving a treatment modality for treatment of an
infectious disease. Such treatment modalities are known in the art
and include but are not limited to antibiotics, antivirals,
antifungals as well as biological and immunotherapeutic agents.
Preferably the HSP preparation comprises HSP-peptide complexes
which display the antigenicity of an agent of the infectious
disease. Preferably the .alpha.2M preparation comprises
.alpha.2M-peptide complexes which display the antigenicity of an
agent of the infectious disease. In a specific embodiment, the
outcome of a treatment of a type of infectious disease in a subject
receiving a non-vaccine therapeutic modality is improved by
administering HSP-peptide complexes comprising an HSP complexed to
a peptide that displays the antigenicity of an antigen of an agent
of said type of infectious disease. Preferably, the HSP-peptide
complexes are not present in admixture with HSP or .alpha.2M that
is not complexed to a peptide that displays the antigenicity of an
antigen of an agent of the same infectious disease. (See
International Application No. PCT/US01/28840, filed Sep. 15, 2001).
In one embodiment, the HSP preparation is administered prior to
administration of the therapeutic modality. In another embodiment,
the therapeutic modality is administered prior to administration of
the HSP preparation. In another specific embodiment, the outcome of
a treatment of a type of infectious disease in a subject receiving
a non-vaccine therapeutic modality is improved by administering
.alpha.2M-peptide complexes comprising an .alpha.2M complexed to a
peptide that displays the antigenicity of an antigen of an agent of
said type of infectious disease. Preferably, the .alpha.2M-peptide
complexes are not present in admixture with HSP or .alpha.2M that
is not complexed to a peptide that displays the antigenicity of an
antigen of an agent of the same infectious disease. Preferably, the
.alpha.2M preparation is administered prior to administration of
the therapeutic modality.
[0061] In another embodiment, the methods comprise the
administration of either an HSP preparation or an .alpha.2M
preparation, preferably a purified HSP preparation or a purified
.alpha.2M preparation, to a subject receiving a treatment modality
for treatment of cancer. Such treatment modalities include but are
not limited to anti-cancer therapies such as chemotherapies and
radiation therapies as well as hormonal therapies, biological
therapies and immunotherapies. In the methods of the invention the
anti-cancer agents that can be used include but are not limited to
cytotoxic agents, antimitotic agents, tubulin stabilizing agents,
microtubule formation inhibiting agents, topoisomerase active
agents, alkylating agents, DNA interactive agents, antimetabolites,
RNA/DNA antimetabolites, and DNA antimetabolites. Preferably, the
anti-cancer agent is a chemotherapeutic agent. Preferably the HSP
preparation or .alpha.2M preparation is administered to a subject
receiving a chemotherapy or radiation therapy for treatment of
cancer. Preferably the HSP preparation comprises HSP-peptide
complexes which display the antigenicity of the type of cancer
being treated. Preferably where the preparation is an .alpha.2M
preparation, the .alpha.2M preparation comprises .alpha.2M-peptide
complexes which display the antigenicity of the type of cancer
being treated. Accordingly, in preferred embodiments, the invention
provides methods for improving the outcome of cancer treatment in a
subject receiving a therapeutic modality which is not a vaccine
using HSP-peptide complexes comprising an HSP complexed to a
peptide that displays the antigenicity of a tumor specific antigen
or tumor associated antigen of a type of cancer or using
.alpha.2M-peptide complexes comprising an .alpha.2M complexed to a
peptide that displays the antigenicity of a tumor specific antigen
or tumor associated antigen of a type of cancer. In certain
preferred embodiments, such HSP-peptide complexes and
.alpha.2M-peptide complexes are not diluted with either HSP or
.alpha.2M that is not complexed to a peptide that displays the
antigenicity of an antigen of the same type of cancer. Preferably,
the HSP preparation or .alpha.2M preparation is administered prior
to administration of the therapeutic modality.
[0062] In a specific embodiment, an HSP preparation is administered
to a subject receiving a chemotherapeutic agent for treatment of
cancer. In another preferred embodiment, an .alpha.2M preparation
is administered to a subject receiving a chemotherapeutic agent for
treatment of cancer. Such chemotherapeutic agents are known in the
art and include but are not limited to: methotrexate, taxol,
mercaptopurine, thioguanine, hydroxyurea, cytarabine,
cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,
mitomycin, dacarbazine, procarbizine, etoposides, campathecins,
bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin,
plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine,
vinorelbine, paclitaxel, and docetaxel, doxorubicin, epirubicin,
5-fluorouracil, taxanes such as docetaxel and paclitaxel,
leucovorin, levamisole, irinotecan, estramustine, etoposide,
nitrosoureas such as carmustine and lomustine, vinca alkaloids,
platinum compounds, mitomycin, gemcitabine, hexamethylmelamine,
topotecan, tyrosine kinase inhibitors, tyrphostins, Gleevec.TM.
(imatinib mesylate), herbimycin A, genistein, erbstatin, and
lavendustin A. In a preferred embodiment, the chemotherapeutic
agent is Gleevec.TM. (imatinib mesylate).
[0063] In other embodiments, suitable chemotherapeutics include,
but are not limited to, methotrexate, taxol, L-asparaginase,
mercaptopurine, thioguanine, hydroxyurea, cytarabine,
cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,
mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards,
cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan,
camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin,
dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine,
vincristine, vinorelbine, paclitaxel, and docetaxel. In a preferred
embodiment, the anti-cancer agent can be, but is not limited to, a
drug listed in Table 1.
TABLE-US-00001 TABLE 1 Alkylating agents Nitrogen mustards:
Cyclophosphamide Ifosfamide Trofosfamide Chlorambucil Nitrosoureas:
Carmustine (BCNU) Lomustine (CCNU) Alkylsulphonates: Busulfan
Treosulfan Triazenes: Dacarbazine Platinum containing Cisplatin
compounds: Carboplatin Aroplatin Oxaliplatin Plant Alkaloids Vinca
alkaloids: Vincristine Vinblastine Vindesine Vinorelbine Taxoids:
Paclitaxel Docetaxel DNA Topoisomerase Inhibitors Epipodophyllins:
Etoposide Teniposide Topotecan 9-aminocamptothecin Camptothecin
Crisnatol mitomycins: Mitomycin C Anti-metabolites Anti-folates:
DHFR inhibitors: Methotrexate Trimetrexate IMP dehydrogenase
Mycophenolic acid Inhibitors: Tiazofurin Ribavirin EICAR
Ribonuclotide reductase Hydroxyurea Inhibitors: Deferoxamine
Pyrimidine analogs: Uracil analogs: 5-Fluorouracil Floxuridine
Doxifluridine Ratitrexed Cytosine analogs: Cytarabine (ara C)
Cytosine arabinoside Fludarabine Purine analogs: Mercaptopurine
Thioguanine DNA Antimetabolites: 3-HP 2'-deoxy-5-fluorouridine 5-HP
alpha-TGDR aphidicolin glycinate ara-C 5-aza-2'-deoxycytidine
beta-TGDR cyclocytidine guanazole inosine glycodialdehyde macebecin
II pyrazoloimidazole Hormonal therapies: Receptor antagonists:
Anti-estrogen: Tamoxifen Raloxifene Megestrol LHRH agonists:
Goserelin Leuprolide acetate Anti-androgens: Flutamide Bicalutamide
Retinoids/Deltoids Cis-retinoic acid Vitamin A derivative:
All-trans retinoic acid (ATRA-IV) Vitamin D3 analogs: EB 1089 CB
1093 KH 1060 Photodynamic therapies: Vertoporfin (BPD-MA)
Phthalocyanine Photosensitizer Pc4 Demethoxy-hypocrellin A
(2BA-2-DMHA) Cytokines: Interferon-.alpha. Interferon-.gamma. Tumor
necrosis factor Angiogenesis Inhibitors: Angiostatin (plasminogen
fragment) antiangiogenic antithrombin III Angiozyme ABT-627 Bay
12-9566 Benefin Bevacizumab BMS-275291 cartilage-derived inhibitor
(CDI) CAI CD59 complement fragment CEP-7055 Col 3 Combretastatin
A-4 Endostatin (collagen XVIII fragment) Fibronectin fragment
Gro-beta Halofuginone Heparinases Heparin hexasaccharide fragment
HMV833 Human chorionic gonadotropin (hCG) IM-862 Interferon
alpha/beta/gamma Interferon inducible protein (IP-10)
Interleukin-12 Kringle 5 (plasminogen fragment) Marimastat
Metalloproteinase inhibitors (TIMPs) 2-Methoxyestradiol MMI 270
(CGS 27023A) MoAb IMC-1C11 Neovastat NM-3 Panzem PI-88 Placental
ribonuclease inhibitor Plasminogen activator inhibitor Platelet
factor-4 (PF4) Prinomastat Prolactin 16 kD fragment
Proliferin-related protein (PRP) PTK 787/ZK 222594 Retinoids
Solimastat Squalamine SS 3304 SU 5416 SU6668 SU11248
Tetrahydrocortisol-S tetrathiomolybdate thalidomide
Thrombospondin-1 (TSP-1) TNP-470 Transforming growth factor-beta
(TGF-b) Vasculostatin Vasostatin (calreticulin fragment) ZD6126 ZD
6474 farnesyl transferase inhibitors (FTI) bisphosphonates
Antimitotic agents: allocolchicine Halichondrin B colchicine
colchicine derivative dolstatin 10 maytansine rhizoxin
thiocolchicine trityl cysteine Others: Isoprenylation inhibitors:
Dopaminergic neurotoxins: 1-methyl-4-phenylpyridinium ion Cell
cycle inhibitors: Staurosporine Actinomycins: Actinomycin D
Dactinomycin Bleomycins: Bleomycin A2 Bleomycin B2 Peplomycin
Anthracyclines: Daunorubicin Doxorubicin (adriamycin) Idarubicin
Epirubicin Pirarubicin Zorubicin Mitoxantrone MDR inhibitors:
Verapamil Ca.sup.2+ATPase inhibitors: Thapsigargin
[0064] Additional anti-cancer agents that may be used in the
methods of the present invention include, but are not limited to:
acivicin; aclarubicin; acodazole hydrochloride; acronine;
adozelesin; aldesleukin; altretamine; ambomycin; ametantrone
acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin;
asparaginase; asperlin; azacitidine; azetepa; azotomycin;
batimastat; benzodepa; bicalutamide; bisantrene hydrochloride;
bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar
sodium; bropirimine; busulfan; cactinomycin; calusterone;
caracemide; carbetimer; carboplatin; carmustine; carubicin
hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;
cisplatin; cladribine; crisnatol mesylate; cyclophosphamide;
cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride;
decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;
diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;
droloxifene; droloxifene citrate; dromostanolone propionate;
duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin;
enloplatin; enpromate; epipropidine; epirubicin hydrochloride;
erbulozole; esorubicin hydrochloride; estramustine; estramustine
phosphate sodium; etanidazole; etoposide; etoposide phosphate;
etoprine; fadrozole hydrochloride; fazarabine; fenretinide;
floxuridine; fludarabine phosphate; fluorouracil; flurocitabine;
fosquidone; fostriecin sodium; gemcitabine; gemcitabine
hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;
ilmofosine; interleukin II (including recombinant interleukin U, or
rIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;
interferon alfa-n3; interferon beta-I a; interferon gamma-I b;
iproplatin; irinotecan hydrochloride; lanreotide acetate;
letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol
sodium; lomustine; losoxantrone hydrochloride; masoprocol;
maytansine; mechlorethamine hydrochloride; megestrol acetate;
melengestrol acetate; melphalan; menogaril; mercaptopurine;
methotrexate; methotrexate sodium; metoprine; meturedepa;
mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;
mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;
mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;
paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin
sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone
hydrochloride; plicamycin; plomestane; porfimer sodium;
porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;
puromycin hydrochloride; pyrazofurin; riboprine; rogletimide;
safingol; safingol hydrochloride; semustine; simtrazene; sparfosate
sodium; sparsomycin; spirogermanium hydrochloride; spiromustine;
spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin;
tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin;
teniposide; teroxirone; testolactone; thiamiprine; thioguanine;
thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone
acetate; triciribine phosphate; trimetrexate; trimetrexate
glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard;
uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine
sulfate; vindesine; vindesine sulfate; vinepidine sulfate;
vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;
vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;
zinostatin; zorubicin hydrochloride.
[0065] Other anti-cancer drugs that can be used include, but are
not limited to: 20-epi-1,25 dihydroxyvitamin 133; 5-ethynyluracil;
abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin;
aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox;
amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide;
anastrozole; andrographolide; angiogenesis inhibitors; antagonist
D; antagonist G; antarelix; anti-dorsalizing morphogenetic
protein-1; antiandrogen, prostatic carcinoma; antiestrogen;
antineoplaston; antisense oligonucleotides; aphidicolin glycinate;
apoptosis gene modulators; apoptosis regulators; apurinic acid;
ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;
atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;
azasetron; azatoxin; azatyrosine; baccatin III derivatives;
balanol; batimastat; BCR/ABL antagonists; benzochlorins;
benzoylstaurosporine; beta lactam derivatives; beta-alethine;
betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide;
bisantrene; bisaziridinylspermine; bisnafide; bistratene A;
bizelesin; breflate; bropirimine; budotitane; buthionine
sulfoximine; calcipotriol; calphostin C; camptothecin derivatives;
canarypox IL-2; capecitabine; carboxamide-amino-triazole;
carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived
inhibitor; carzelesin; casein kinase inhibitors (ICOS);
castanospermine; cecropin B; cetrorelix; chlorins;
chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl
spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;
droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;
edelfosine; edrecolomab; eflornithine; elemene; emitefur;
epirubicin; epristeride; estramustine analogue; estrogen agonists;
estrogen antagonists; etanidazole; etoposide phosphate; exemestane;
fadrozole; fazarabine; fenretinide; filgrastim; finasteride;
flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
glutathione inhibitors; hepsulfam; heregulin; hexamethylene
bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;
idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;
immunostimulant peptides; insulin-like growth factor-1 receptor
inhibitor; interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;
isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia
inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;
lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;
masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase
inhibitors; menogaril; merbarone; meterelin; methioninase;
metoclopramide; MIF inhibitor; mifepristone; miltefosine;
mirimostim; mismatched double stranded RNA; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
monoclonal antibody, human chorionic gonadotrophin; monophosphoryl
lipid A+myobacterium cell wall sk; mopidamol; multiple drug
resistance gene inhibitor; multiple tumor suppressor 1-based
therapy; mustard anti-cancer agent; mycaperoxide B; mycobacterial
cell wall extract; myriaporone; N-acetyldinaline; N-substituted
benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin;
naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid;
neutral endopeptidase; nilutamide; nisamycin; nitric oxide
modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine;
octreotide; okicenone; oligonucleotides; onapristone; ondansetron;
ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;
oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;
paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic
acid; panaxytriol; panomifene; parabactin; pazelliptine;
pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;
pentrozole; perflubron; perfosfamide; perillyl alcohol;
phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil;
pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A;
placetin B; plasminogen activator inhibitor; platinum complex;
platinum compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;
roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU;
sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence
derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal transduction modulators; single chain antigen
binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium
phenylacetate; solverol; somatomedin binding protein; sonermin;
sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stem cell inhibitor, stem-cell division
inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;
superactive vasoactive intestinal peptide antagonist; suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;
tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;
tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;
temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;
thaliblastine; thiocoraline; thrombopoietin; thrombopoietin
mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;
thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;
titanocene bichloride; topsentin; toremifene; totipotent stem cell
factor; translation inhibitors; tretinoin; triacetyluridine;
triciribine; trimetrexate; triptorelin; tropisetron; turosteride;
tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;
urogenital sinus-derived growth inhibitory factor; urokinase
receptor antagonists; vapreotide; variolin B; vector system,
erythrocyte gene therapy; velaresol; veramine; verdins;
verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;
zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
Preferred chemotherapeutics of the invention include Gleevec.TM.
(imatinib mesylate) and other tyrosine kinase inhibitors.
[0066] In preferred embodiments, each of the methods above comprise
administering either an HSP preparation or an .alpha.2M
preparation, preferably a purified HSP preparation or a purified
.alpha.2M preparation, to a subject receiving a drug of the
2-phenylaminopyrimidine class for treatment of cancer. More
preferably, the subject is receiving Gleevec.TM. (i.e., imatinib
mesylate) for treatment of cancer.
[0067] In another preferred embodiment, an HSP preparation or an
.alpha.2M preparation is administered to a subject receiving
radiation therapy for treatment of cancer. For radiation treatment,
the radiation can be gamma rays or X-rays. The methods encompass
treatment of cancer comprising radiation therapy, such as
external-beam radiation therapy, interstitial implantation of
radioisotopes (I-125, palladium, iridium), radioisotopes such as
strontium-89, thoracic radiation therapy, intraperitoneal P-32
radiation therapy, and/or total abdominal and pelvic radiation
therapy. For a general overview of radiation therapy, see Hellman,
Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th
edition, 2001, DeVita et al., eds., J.B. Lippencott Company,
Philadelphia. In preferred embodiments, the radiation treatment is
administered as external beam radiation or teletherapy wherein the
radiation is directed from a remote source. In various preferred
embodiments, the radiation treatment is administered as internal
therapy or brachytherapy wherein a radioactive source is placed
inside the body close to cancer cells or a tumor mass.
[0068] In another embodiment, the each of the above methods
comprise the administration of HSP preparation, preferably a
purified HSP preparation, to a subject receiving a combination of
treatment modalities for the treatment of cancer. In another
embodiment, the each of the above methods comprise the
administration of an .alpha.2M preparation, preferably a purified
.alpha.2M preparation, to a subject receiving a combination of
treatment modalities for the treatment of cancer. Preferably the
HSP preparation and .alpha.2M preparation each comprises
HSP-peptide complexes and .alpha.2M-peptide complexes,
respectively, which display the antigenicity of the type of cancer
being treated. In one such embodiment, HSP preparation is
administered to a subject receiving a chemotherapy in combination
with a biological therapy, preferably a cytokine. In another such
embodiment, an .alpha.2M preparation is administered to a subject
receiving a chemotherapy in combination with a biological therapy,
preferably a cytokine. In various embodiments, the cytokine is
selected from the group consisting of IL-1.alpha., IL-1.beta.,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IFN.alpha., IFN.beta., IFN.gamma., TNF.alpha., TNF.beta.,
G-CSF, GM-CSF, TGF-.beta., IL-15, IL-18, GM-CSF, INF-.gamma., SLC,
endothelial monocyte activating protein-2 (EMAP2), MIP-3.alpha.,
MIP-3.beta., or an MHC gene, such as HLA-B7. Additionally, other
exemplary cytokines include other members of the TNF family,
including but not limited to TNF-.alpha.-related apoptosis-inducing
ligand (TRAIL), TNF-.alpha.-related activation-induced cytokine
(TRANCE), TNF-.alpha.-related weak inducer of apoptosis (TWEAK),
CD40 ligand (CD40L), LT-.alpha., LT-.beta., OX4OL, CD4OL, FasL,
CD27L, CD30L, 4-1BBL, APRIL, LIGHT, TL1, TNFSF16, TNFSF17, and
AITR-L, or a functional portion thereof. See, e.g., Kwon et al.,
1999, Cum Opin. Immunol. 11:340-345 for a general review of the TNF
family. Preferably, the HSP preparation is administered prior to
the treatment modalities.
[0069] In a specific embodiment, a purified HSP preparation is
administered to a subject receiving cyclophosphamide in combination
with IL-12 for treatment of cancer. In another specific embodiment,
a purified .alpha.2M preparation is administered to a subject
receiving cyclophosphamide in combination with IL-12 for treatment
of cancer.
[0070] In another specific embodiment, the chemotherapeutic is a
tyrosine kinase inhibitor, the HSP preparation is obtained from the
cancer subject being treated, and the chemotherapy is administered
prior to administration of the HSP preparation. In another specific
embodiment, the anti-cancer agent is the chemotherapeutic
Gleevec.TM. (imatinib mesylate), the HSP preparation comprises
hsp70 obtained from the cancer subject being treated, and the
chemotherapeutic is administered prior to administration of the HSP
preparation. In another specific embodiment, the HSP preparation
comprises hsp70-peptide complexes obtained from the cancer subject
being treated. Another specific embodiment encompasses a method for
treating CML in a subject receiving about 400 mg to 800 mg of
imatinib mesylate daily comprising administering a heat shock
protein preparation to said subject, wherein said heat shock
protein preparation comprises hsp70 peptide complexes. In preferred
embodiments, the heat shock protein preparation is administered
once a week and the heat shock protein preparation comprises
hsp70-peptide complexes obtained from said subject.
[0071] In certain specific embodiments, an HSP preparation is
administered to a subject already receiving Gleevec.TM. (e.g.,
400-800 mg daily in capsule form, 400-600 mg doses administered
once daily, or 800 mg dose administered daily in two doses of 400
mg each). In such embodiments, an HSP/.alpha.2M preparation is
initially administered to a subject who has already been receiving
Gleevec.TM. in the absence of HSP/.alpha.2M preparation 2 days, 2
days to 1 week, 1 week to 1 month, 1 month to 6 months, 6 months to
1 year prior to administration of HSP/.alpha.2M preparation in
addition to Gleevec.TM.. In a specific embodiment, an HSP/.alpha.2M
preparation is administered to a subject wherein the subject showed
resistance to treatment with Gleevec.TM. alone.
[0072] In other embodiments, an HSP/.alpha.2M preparation is
initially administered to a subject concurrently with the initial
administration of Gleevec.TM..
[0073] In yet other specific embodiments, Gleevec.TM. (e.g.,
400-800 mg daily in capsule form) is administered to a subject
already receiving treatment comprising administration of an
HSP/.alpha.2M preparation. In such embodiments, Gleevec.TM. is
initially administered to a subject who has already been receiving
an HSP/.alpha.2M preparation in the absence of Gleevec.TM. 2 days,
2 days to 1 week, 1 week to 1 month, 1 month to 6 months, 6 months
to 1 year prior to administration of Gleevec.TM. in addition to
administration of an HSP/.alpha.2M preparation.
[0074] In a specific embodiment, Gleevec.TM. is administered
orally. In another specific embodiment, the HSP preparation is
administered intradermally.
[0075] In each of the methods contemplated above, the patient, by
way of example, receives 50 mg to 100 mg, 100 mg to 200 mg, 200 mg
to 300 mg, 300 mg to 400 mg, 400 mg to 500 mg, 500 mg to 600 mg,
600 mg to 700 mg, 700 mg to 800 mg, 800 mg to 900 mg, or 900 mg to
1000 mg of Gleevec.TM. daily. In certain embodiments, the total
daily dose is administered to a subject as two daily doses of 25 mg
to 50 mg, 50 mg to 100 mg, 100 mg to 200 mg, 200 mg to 300 mg, 300
mg to 400 mg, or 400 mg to 500 mg.
[0076] Other treatment modalities contemplated include but are not
limited to antiviral agents known in the art. Such antiviral agents
include but are not limited to: ribavirin, rifampicin, AZT, ddI,
ddC, acyclovir and ganciclovir.
[0077] Also encompassed by the invention are therapeutic modalities
that are antibiotic agents known in the art including but not
limited to: aminoglycoside antibiotics apramycin, arbekacin,
bambermycins, butirosin, dibekacin, neomycin, neomycin,
undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, and
spectinomycin), amphenicol antibiotics (e.g., azidamfenicol,
chloramphenicol, florfenicol, and thiamphenicol), ansamycin
antibiotics (e.g., rifamide and rifampin), carbacephems (e.g.,
loracarbef), carbapenems (e.g., biapenem and imipenem),
cephalosporins (e.g., cefaclor, cefadroxil, cefamandole,
cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, and
cefpirome), cephamycins (e.g., cefbuperazone, cefmetazole, and
cefminox), monobactams (e.g., aztreonam, carumonam, and tigemonam),
oxacephems (e.g., flomoxef, and moxalactam), penicillins (e.g.,
amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin,
benzylpenicillinic acid, benzylpenicillin sodium, epicillin,
fenbenicillin, floxacillin, penamecillin, penethamate hydriodide,
penicillin o-benethamine, penicillin 0, penicillin V, penicillin V
benzathine, penicillin V hydrabamine, penimepicycline, and
phencihicillin potassium), lincosamides (e.g., clindamycin, and
lincomycin), macrolides (e.g., azithromycin, carbomycin,
clarithomycin, dirithromycin, erythromycin, and erythromycin
acistrate), amphomycin, bacitracin, capreomycin, colistin,
enduracidin, enviomycin, tetracyclines (e.g., apicycline,
chlortetracycline, clomocycline, and demeclocycline),
2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans (e.g.,
furaltadone, and furazolium chloride), quinolones and analogs
thereof (e.g., cinoxacin, ciprofloxacin, clinafloxacin, flumequine,
and grepagloxacin), sulfonamides (e.g., acetyl
sulfamethoxypyrazine, benzylsulfamide, noprylsulfamide,
phthalylsulfacetamide, sulfachrysoidine, and sulfacytine), sulfones
(e.g., diathymosulfone, glucosulfone sodium, and solasulfone),
cycloserine, mupirocin and tuberin.
[0078] Also encompassed by the invention are therapeutic modalities
that are antifungal agents and known in the art and include but are
not limited to: polyenes (e.g., amphotericin b, candicidin,
mepartricin, natamycin, and nystatin), allylamines (e.g.,
butenafine, and naftifine), imidazoles (e.g., bifonazole,
butoconazole, chlordantoin, flutrimazole, isoconazole,
ketoconazole, and lanoconazole), thiocarbamates (e.g., tolciclate,
tolindate, and tolnaftate), triazoles fluconazole, itraconazole,
saperconazole, and terconazole), bromosalicylchloranilide,
buclosamide, calcium propionate, chlorphenesin, ciclopirox,
azaserine, griseofulvin, oligomycins, neomycin undecylenate,
pyrrolnitrin, siccanin, tubercidin, and viridin.
[0079] In another embodiment, the above methods are useful for the
prevention of cancer or infectious disease. In a specific
embodiment, an HSP preparation is administered in conjunction with
a non-vaccine treatment modality to a subject to reduce the risk of
acquiring a type of cancer or an infectious disease. In other
specific embodiments, the methods encompass administration of an
HSP preparation with administration of a non-vaccine treatment
modality as a preventative measure to a subject having a genetic or
non-genetic predisposition to a cancer or infectious disease or to
a subject facing exposure to an agent of an infectious disease. In
further embodiments, the invention also provides that each of the
foregoing embodiments also can be applicable wherein an .alpha.2M
preparation is administered in conjunction with a non-vaccine
treatment modality.
[0080] The methods and compositions of the invention are useful not
only in untreated patients, but are also useful in the treatment of
patients partially or completely un-responsive to the therapeutic
modality in the absence of the HSP/.alpha.2M preparation or to the
HSP/.alpha.2M preparation in the absence of the therapeutic
modality. In various embodiments, the invention provides methods
and compositions useful in the treatment or prevention of diseases
and disorders in patients that have been shown to be or may be
refractory or non-responsive to therapies comprising the
administration of either or both the HSP/.alpha.2M preparation or
the therapeutic modality. The invention also includes methods and
compositions comprising administration of the HSP/.alpha.2M
preparation and the therapeutic modality to patients that have
previously received and/or are concurrently receiving other forms
of medical therapy.
[0081] The HSP preparation used in the methods and compositions of
the invention is preferably purified, and can include free HSP not
bound to any molecule, and molecular complexes of HSP with another
molecule, such as a peptide. An HSP-peptide complex comprises an
HSP covalently or noncovalently attached to a peptide. The methods
of the invention may or may not require covalent or noncovalent
attachment of an HSP to any specific antigens or antigenic peptides
prior to administration to a subject. Although, the peptide(s) may
be unrelated to the infectious disease or disorder or particular
cancer being treated, in preferred embodiments, the HSP preparation
comprises complexes which display the antigenicity of an antigen of
the agent of infectious disease or of a tumor specific antigen or
tumor associated antigen of the type of cancer being treated,
respectively. More preferably, for the treatment of infectious
disease, the HSP preparation comprises noncovalent HSP-peptide
complexes isolated from a cell infected with an infectious agent
(or non-infectious variant thereof displaying the antigenicity
thereof) that causes the infectious disease. More preferably, for
treatment of a type of cancer, the HSP preparation comprises
noncovalent HSP-peptide complexes isolated from cancerous tissue of
said type of cancer or a metastasis thereof, which can be from the
patient (autologous) or not (allogeneic). Accordingly, for the
purposes of this invention, an HSP preparation is a composition
comprising HSPs whether unbound or bound to other molecules (e.g.,
peptides). The HSP is preferably purified. An HSP preparation may
include crude cell lysate comprising HSP, the amount of lysate
corresponding to between 100 to 10.sup.8 cell equivalents. HSPs can
be conveniently purified from most cellular sources as a population
of complexes of different peptides non-covalently bound to HSPs.
The HSPs can be separated from the non-covalently bound peptides by
exposure to low pH and/or adenosine triphosphate, or other methods
known in the art.
[0082] The .alpha.2M preparation used in the methods and
compositions of the invention is preferably purified, and can
include free .alpha.2M not bound to any molecule, and molecular
complexes of .alpha.2M with another molecule, such as a peptide. An
.alpha.2M-peptide complex comprises an .alpha.2M covalently or
noncovalently attached to a peptide. The methods of the invention
may or may not require covalent or noncovalent attachment of an
.alpha.2M to any specific antigens or antigenic peptides prior to
administration to a subject. Although, the peptide(s) may be
unrelated to the infectious disease or disorder or particular
cancer being treated, in preferred embodiments, the .alpha.2M
preparation comprises complexes which display the antigenicity of
an antigen of the agent of infectious disease or of a tumor
specific antigen or tumor associated antigen of the type of cancer
being treated, respectively. More preferably, for the treatment of
infectious disease, the .alpha.2M preparation comprises noncovalent
.alpha.2M-peptide complexes isolated from a cell infected with an
infectious agent (or non-infectious variant thereof displaying the
antigenicity thereof) that causes the infectious disease. More
preferably, for treatment of a type of cancer, the .alpha.2M
preparation comprises noncovalent .alpha.2M-peptide complexes
isolated from cancerous tissue of said type of cancer or a
metastasis thereof, which can be from the patient (autologous) or
not (allogeneic). Accordingly, for the purposes of this invention,
an .alpha.2M preparation is a composition comprising .alpha.2M
whether unbound or bound to other molecules (e.g., peptides). The
.alpha.2M is preferably purified. An .alpha.2M preparation may
include crude cell lysate comprising .alpha.2M, the amount of
lysate corresponding to between 100 to 10.sup.8 cell equivalents.
.alpha.2M s can be conveniently purified from most cellular sources
as a population of complexes of different peptides non-covalently
bound to .alpha.2Ms. The .alpha.2M can be separated from the
non-covalently bound peptides by exposure to low pH and/or
adenosine triphosphate, or other methods known in the art.
[0083] In various embodiments, the source of the HSP and the
.alpha.2M is preferably an eukaryote, more preferably a mammal, and
most preferably a human. Accordingly, the HSP preparation used by
the methods of the invention includes eukaryotic HSPs, mammalian
HSPs and human HSPs. The .alpha.2M preparation includes eukaryotic
.alpha.2M, mammalian .alpha.2M and human .alpha.2M. The eukaryotic
source from which the HSP preparation or .alpha.2M preparation is
derived and the subject receiving the HSP preparation or the
.alpha.2M preparation, respectively, are preferably the same
species.
[0084] In one embodiment, the specific immunogenicity of the HSP
preparation derives from the peptide complexed to a heat shock
protein. Accordingly, in various embodiments, the HSP preparation
comprises heat shock protein peptide complexes wherein the heat
shock proteins are complexed to peptides derived from a specific
antigen source. In a preferred embodiment, the HSP protein
preparation comprises heat shock protein-peptide complexes that are
autologous. In another preferred embodiment, the HSP preparation
comprises heat shock proteins complexed to antigenic peptides of
the cancer cells from which they are derived. In specific
embodiments, the antigen is a tumor specific antigen (i.e., only
expressed in the tumor cells). In other specific embodiments, the
antigen is a tumor associated antigen (i.e., relatively
overexpressed in the tumor cells). In yet another preferred
embodiment, the HSP preparation comprises heat shock proteins
complexed to antigenic peptides of the infected cells from which
they are derived.
[0085] In another embodiment, the specific immunogenicity of the
.alpha.2M preparation derives from the peptide complexed to an
.alpha.2M. Accordingly, in various embodiments, the .alpha.2M
preparation comprises .alpha.2M peptide complexes wherein the
.alpha.2M are complexed to peptides derived from a specific antigen
source. In a preferred embodiment, the .alpha.2M protein
preparation comprises .alpha.2M-peptide complexes that are
autologous. In another preferred embodiment, the .alpha.2M
preparation comprises .alpha.2M complexed to antigenic peptides of
the cancer cells from which they are derived. In other specific
embodiments, the antigen is a tumor associated antigenic (i.e.,
relatively overexpressed in the tumor cells). In yet another
preferred embodiment, the .alpha.2M preparation comprises .alpha.2M
complexed to antigenic peptides of the infected cells from which
they are derived.
[0086] In various specific embodiments, the above methods comprise
the administration of HSP preparation or .alpha.2M preparation to a
subject treated with a treatment modality wherein the treatment
modality administered alone is not clinically adequate to treat the
subject such that the subject needs additional effective therapy,
e.g., a subject is unresponsive to a treatment modality without
administering HSP preparation or .alpha.2M preparation. Included in
such embodiments are methods comprising administering HSP
preparation or .alpha.2M preparation to a subject receiving a
treatment modality wherein said subject has responded to therapy
yet suffers from side effects, relapse, develops resistance, etc.
Such a subject might be non-responsive or refractory to treatment
with the treatment modality alone. The embodiments provide that the
methods of the invention comprising administration of HSP
preparation to a subject refractory to a treatment modality alone
can improve the therapeutic effectiveness of the treatment modality
when administered as contemplated by the methods of the invention.
The methods of the invention comprising administration of an
.alpha.2M preparation to a subject refractory to a treatment
modality alone can also improve the therapeutic effectiveness of
the treatment modality when administered as contemplated by the
methods of the invention.
[0087] In a specific embodiment, an HSP preparation is administered
to a subject receiving a treatment modality for the treatment of
cancer wherein the subject may be non-responsive or refractory to
treatment with the treatment modality alone, i.e., at least some
significant portion of cancer cells are not killed or their cell
division is not arrested. The determination of the effectiveness of
a treatment modality can be assayed in vivo or in vitro using
methods known in the art. Art-accepted meanings of refractory are
well known in the context of cancer. In one embodiment, a cancer is
refractory or non-responsive where the number of cancer cells has
not been significantly reduced, or has increased. In a preferred
embodiment, an HSP preparation that displays the antigenicity of a
type of cancer is administered to a subject non-responsive to
administration of a treatment modality alone, wherein the
administration of HSP preparation improves the effectiveness of the
treatment modality. Among these subjects being treated are those
receiving chemotherapy or radiation therapy.
[0088] In a specific embodiment, an .alpha.2M preparation is
administered to a subject receiving a treatment modality for the
treatment of cancer wherein the subject may be non-responsive or
refractory to treatment with the treatment modality alone, i.e., at
least some significant portion of cancer cells are not killed or
their cell division is not arrested. The determination of the
effectiveness of a treatment modality can be assayed in vivo or in
vitro using methods known in the art. Art-accepted meanings of
refractory are well known in the context of cancer. In one
embodiment, a cancer is refractory or non-responsive where the
number of cancer cells has not been significantly reduced, or has
increased. In a preferred embodiment, an .alpha.2M preparation that
displays the antigenicity of a type of cancer is administered to a
subject non-responsive to administration of a treatment modality
alone, wherein the administration of .alpha.2M preparation improves
the effectiveness of the treatment modality. Among these subjects
being treated are those receiving chemotherapy or radiation
therapy.
[0089] In a specific embodiment, an HSP preparation is administered
to a subject receiving a treatment modality for the treatment of
cancer wherein the subject may experience unwanted or adverse
effects to treatment with the treatment modality alone, e.g., the
treatment modality may be toxic or harmful at its effective dose,
administered alone. Given the invention, the HSP preparation can
improve the therapeutic benefit of the treatment modality such that
the dosage or frequency of administration of the treatment modality
can be lowered when administered in conjunction with HSP
preparation. In a preferred embodiment, an HSP preparation that
displays the antigenicity of a type of cancer is administered to a
subject to reduce or avoid the unwanted or adverse effects of a
treatment modality alone, wherein the administration of HSP
preparation allows lower and/or less frequent doses of the
treatment modality. Among these subjects being treated are those
receiving chemotherapy or radiation therapy.
[0090] In a specific embodiment, an .alpha.2M preparation is
administered to a subject receiving a treatment modality for the
treatment of cancer wherein the subject may experience unwanted or
adverse effects to treatment with the treatment modality alone,
e.g., the treatment modality may be toxic or harmful at its
effective dose, administered alone. Given the invention, the
.alpha.2M preparation can improve the therapeutic benefit of the
treatment modality such that the dosage or frequency of
administration of the treatment modality can be lowered when
administered in conjunction with .alpha.2M preparation. In a
preferred embodiment, an .alpha.2M preparation that displays the
antigenicity of a type of cancer is administered to a subject to
reduce or avoid the unwanted or adverse effects of a treatment
modality alone, wherein the administration of .alpha.2M preparation
allows lower and/or less frequent doses of the treatment modality.
Among these subjects being treated are those receiving chemotherapy
or radiation therapy.
[0091] In a specific embodiment, the HSP preparation is
administered in a sub-optimal amount, e.g., an amount that does not
manifest detectable therapeutic benefits when administered in the
absence of the therapeutic modality, as determined by methods known
in the art. In such methods, the administration of such a
sub-optimal amount of HSP preparation to a subject receiving a
therapeutic modality results in an overall improvement in
effectiveness of treatment. In another specific embodiment, the
.alpha.2M preparation is administered in a sub-optimal amount. In
such methods, the administration of such a sub-optimal amount of
.alpha.2M preparation to a subject receiving a therapeutic modality
results in an overall improvement in effectiveness of
treatment.
[0092] In a preferred embodiment, an HSP preparation is
administered in an amount that does not result in tumor regression
or cancer remission or an amount wherein the cancer cells have not
been significantly reduced or have increased when said HSP
preparation is administered in the absence of the therapeutic
modality. Preferably the HSP preparation comprises HSP-peptide
complexes displaying the antigenicity of the cancer type being
treated. In a preferred embodiment, the sub-optimal amount of HSP
preparation is administered to a subject receiving a treatment
modality whereby the overall effectiveness of treatment is
improved. In another preferred embodiment, an .alpha.2M preparation
is administered in an amount that does not result in tumor
regression or cancer remission or an amount wherein the cancer
cells have not been significantly reduced or have increased when
said .alpha.2M preparation is administered in the absence of the
therapeutic modality. Preferably the .alpha.2M preparation
comprises .alpha.2M-peptide complexes displaying the antigenicity
of the cancer type being treated. In a preferred embodiment, the
sub-optimal amount of .alpha.2M preparation is administered to a
subject receiving a treatment modality whereby the overall
effectiveness of treatment is improved. Among these subjects being
treated with HSP or .alpha.2M preparation are those receiving
chemotherapy or radiation therapy. A sub-optimal amount can be
determined by appropriate animal studies. Such a sub-optimal amount
in humans can be determined by extrapolation from experiments in
animals.
[0093] The HSP preparation or .alpha.2M preparation can be
administered prior to, concurrently with, or subsequent to the
administration of the non-vaccine treatment modality. In one
embodiment, the HSP preparation and therapeutic modality are
administered at exactly the same time. In another embodiment, the
.alpha.2M preparation and therapeutic embodiment are administered
at exactly the same time. In another embodiment the either the HSP,
preparation or the .alpha.2M preparation and treatment modality are
administered in a sequence and within a time interval such that the
HSP preparation and treatment modality can act together to provide
an increased benefit than if they were administered alone or such
that the .alpha.2M preparation and treatment modality can act
together to provide an increased benefit than if they were
administered alone. In another embodiment, the HSP preparation and
treatment modality are administered sufficiently close in time so
as to provide the desired therapeutic outcome. In another
embodiment, the .alpha.2M preparation and treatment modality are
administered sufficiently close in time so as to provide the
desired therapeutic outcome. The HSP or .alpha.2M preparation and
the therapeutic modality can be administered simultaneously or
separately, in any appropriate form and by any suitable route. In
one embodiment, the HSP preparation and treatment modality are
administered by different routes of administration. In an alternate
embodiment, each is administered by the same route of
administration. The HSP preparation can be administered at the same
or different sites, e.g. arm and leg. In another embodiment, the
.alpha.2M preparation and treatment modality are administered by
different routes of administration. Alternatively, each can be
administered by the same route. In addition, each could be
administered at the same or different sites.
[0094] In various embodiments, such as those described above, the
HSP preparation and treatment modality are administered less than 1
hour apart, at about 1 hour apart, 1 hour to 2 hours apart, 2 hours
to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours
apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours
to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours
apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no
more than 24 hours apart or no more than 48 hours apart, or no more
than 1 week or 2 weeks or 1 month or 3 months apart. In other
embodiments, the HSP preparation and treatment modality are
administered 2 to 4 days apart, 4 to 6 days apart, 1 week apart, 1
to 2 weeks apart, 2 to 4 weeks apart, one month apart, 1 to 2
months apart, or 2 or more months apart. In preferred embodiments,
the HSP preparation and treatment modality are administered in a
time frame where both are still active. One skilled in the art
would be able to determine such a time frame by determining the
half life of each administered component. In separate or in the
foregoing embodiments, the HSP preparation and treatment modality
are administered less than 2 weeks, one month, six months, 1 year
or 5 years apart. Preferably, the HSP preparation is administered
prior to the treatment modality. In further embodiments, the
.alpha.2M preparation and treatment modality are administered at
the time intervals and time frames described in each of the above
embodiments. Preferably the .alpha.2M preparation is administered
prior to the treatment modality. Preferably, in each of the above
embodiments, the treatment modality is a combination of a
chemotherapy and cytokine treatment.
[0095] In one embodiment, the treatment modality is administered
daily and the HSP preparation or .alpha.2M preparation is
administered once a week for the first 4 weeks, and then once every
other week thereafter. In one embodiment, the treatment modality is
administered daily and the HSP preparation or .alpha.2M preparation
is administered once a week for the first 8 weeks, and then once
every other week thereafter.
[0096] In one embodiment, two or more components are administered
within the same patient visit. In one embodiment, the .alpha.2M
preparation is administered prior to the administration of the
treatment modality. In an alternate embodiment, the .alpha.2M
preparation is administered subsequent to the administration of the
treatment modality. In one embodiment, the .alpha.2M preparation is
administered prior to the administration of the treatment modality.
In an alternate embodiment, the HSP preparation is administered
subsequent to the administration of the treatment modality.
[0097] In certain embodiments, the HSP preparation or the .alpha.2M
preparation and non-vaccine treatment modality are cyclically
administered to a subject. Cycling therapy involves the
administration of the HSP preparation for a period of time,
followed by the administration of a treatment modality for a period
of time and repeating this sequential administration.
Alternatively, cycling therapy can involve the administration of
.alpha.2M preparation for a period of time, followed by the
administration of a treatment modality for a period of time and
repeating this sequential administration. Cycling therapy can
reduce the development of resistance to one or more of the
therapies, avoid or reduce the side effects of one of the
therapies, and/or improve the efficacy of the treatment. In such
embodiments, the invention contemplates the alternating
administration of an HSP preparation followed by the administration
of a treatment modality 4 to 6 days later, preferable 2 to 4 days,
later, more preferably 1 to 2 days later, wherein such a cycle may
be repeated as many times as desired. The invention also
contemplates the alternating administration of an .alpha.2M
preparation followed by the administration of a treatment modality
4 to 6 days later, preferable 2 to 4 days, later, more preferably 1
to 2 days later, wherein such a cycle may be repeated as many times
as desired.
[0098] In certain embodiments, the HSP preparation and treatment
modality are alternately administered in a cycle of less than 3
weeks, once every two weeks, once every 10 days or once every week.
In other embodiments, the .alpha.2M preparation and treatment
modality are alternately administered in cycles of less than 3
weeks, once every two weeks, once every 10 days or once every week.
In a specific embodiment of the invention, one cycle can comprise
the administration of a chemotherapeutic by infusion over 90
minutes every cycle, 1 hour every cycle, or 45 minutes every cycle.
Each cycle can comprise at least 1 week of rest, at least 2 weeks
of rest, at least 3 weeks of rest. In an embodiment, the number of
cycles administered is from 1 to 12 cycles, more typically from 2
to 10 cycles, and more typically from 2 to 8 cycles.
[0099] In a preferred embodiment, an HSP preparation displaying the
antigenicity of a tumor specific or tumor associated antigen of a
type of cancer is administered to a subject in an amount
ineffective for treating said cancer about 2 weeks to 1 month prior
to receiving combination chemotherapy with cytokine treatment,
wherein treatment effectiveness is greater than the effectiveness
of HSP preparation or combination chemotherapy with cytokine
treatment administered alone. Preferably the subject is human. In a
preferred embodiment, the subject is non-responsive to combination
chemotherapy with cytokine treatment prior to administration of HSP
preparation. In another preferred embodiment, the chemotherapy is
cyclophosphamide, the cytokine is IL-12, and the HSP preparation
comprises gp96-peptide complexes obtained from cancerous tissue of
the subject.
[0100] In particularly preferred embodiment, an .alpha.2M
preparation displaying the antigenicity of a tumor specific or
tumor associated type of cancer is administered to a subject in an
amount ineffective for treating said cancer about 2 weeks to 1
month prior to receiving combination chemotherapy with cytokine
treatment, wherein treatment effectiveness is greater than the
effectiveness of .alpha.2M preparation or combination chemotherapy
with cytokine treatment administered alone. Preferably the subject
is human. In a preferred embodiment, the subject is non-responsive
to combination chemotherapy with cytokine treatment prior to
administration of .alpha.2M preparation. In another preferred
embodiment, the chemotherapy is cyclophosphamide, the cytokine is
IL-12, and the .alpha.2M preparation comprises .alpha.2M-peptide
complexes obtained from cancerous tissue of the subject.
[0101] In specific embodiments, the above methods encompass the
administration of Gleevec.TM. (imatinib mesylate) for treatment of
cancer. In a preferred embodiment, the cancer is CML, the
chemotherapeutic is Gleevec.TM. (imatinib mesylate), and the HSP
preparation comprises hsp70-peptide complexes obtained from the
cancer subject being treated.
[0102] Also encompassed by the invention are methods of treatment
and delivery, pharmaceutical compositions and formulas comprising
administering at least one non-vaccine therapeutic modality and an
HSP preparation or an .alpha.2M preparation and kits comprising
such pharmaceutical compositions.
5.2. Heat Shock Protein Preparations
[0103] Three major families of HSPs have been identified based on
molecular weight. The families have been called hsp60, hsp70 and
hsp90 where the numbers reflect the approximate molecular weight of
the stress proteins in kilodaltons. Many members of these families
were found subsequently to be induced in response to other
stressful stimuli including, but not limited to, nutrient
deprivation, metabolic disruption, oxygen radicals and infection
with intracellular pathogens (See Welch, May 1993, Scientific
American 56-64; Young, 1990, Annu. Rev. Immunol. 8:401-420; Craig,
1993, Science 260:1902-1903; Gething, et al., 1992, Nature
355:33-45; and Lindquist, et al., 1988, Annu. Rev. Genetics
22:631-677). A number of proteins thought to be involved in
chaperoning functions are residents of the endoplasmic reticulum
(ER) lumen and include, for example, protein disulfide isomerase
(PDI; Gething et al., 1992, Nature 355:33-45), calreticulin
(Herbert et al., 1997, J. Cell Biol. 139:613-623), Grp94 or ERp99
(Sorger & Pelham, 1987, J. Mol. Biol. 194:(2) 341-4) which is
related to hsp90, and Grp78 or BiP, which is related to hsp70
(Munro et al., 1986, Cell 46:291-300; Haas & Webl, 1983, Nature
306:387-389). It is contemplated that HSPs belonging to all of
these three families, including fragments of such HSPs, can be used
in the practice of the instant invention. It is also noted that
HSPs include constitutively expressed conserved cellular homologs
of the proteins induced by stress.
[0104] HSPs are also referred to interchangeably herein as stress
proteins and can be selected from among any cellular protein that
satisfies the following criteria. It is a protein whose
intracellular concentration increases when a cell is exposed to a
stressful stimuli, it is capable of binding other proteins or
peptides, it is capable of releasing the bound proteins or peptides
in the presence of adenosine triphosphate (ATP) or low pH, and it
is a protein showing at least 35% homology with any cellular
protein having any of the above properties.
[0105] Heat shock proteins are among the most highly conserved
proteins in existence. For example, DnaK, the hsp70 from E. coli
has about 50% amino acid sequence identity with hsp70 proteins from
excoriates (Bardwell, et al., 1984, Proc. Natl. Acad. Sci.
81:848-852). The hsp60 and hsp90 families also show similarly high
levels of intra families conservation (Hickey, et al., 1989, Mol.
Cell. Biol. 9:2615-2626; Jindal, 1989, Mol. Cell. Biol.
9:2279-2283). In addition, it has been discovered that the hsp60,
hsp70 and hsp90 families are composed of proteins that are related
to the stress proteins in sequence, for example, having greater
than 35% amino acid identity, but whose expression levels are not
altered by stress. Therefore it is contemplated that stress
proteins/HSPs include other proteins, muteins, analogs, and
variants thereof having at least 35% to 55%, preferably 55% to 75%,
and most preferably 75% to 85% amino acid identity with members of
the three families whose expression levels in a cell are enhanced
in response to a stressful stimulus. The purification of stress
proteins belonging to these three families is described below.
[0106] In addition, HSPs have been found to have immunological and
antigenic properties. HSPs are now understood to play an essential
role in immune regulation. For instance, prior experiments have
demonstrated that HSPs stimulate strong and long-lasting specific
immune responses against antigenic peptides that have been
covalently or noncovalently attached to the HSPs. By utilizing a
specific peptide, the immune response generated is "specific" or
targeted to that peptide.
[0107] Where HSP-peptide complexes are used in conjunction with
administration of a non-vaccine treatment modality, preferably, the
peptides are antigenic or relevant to the condition. In particular
preferred embodiments, it is contemplated that the therapeutic
outcome of a treatment modality administered to a subject with a
particular type of cancer is improved by the administration of an
HSP-peptide complex wherein the peptide displays the antigenicity
of an antigen of that type of cancer.
[0108] In the present invention, an HSP preparation can include but
not be limited to unbound hsp70, hsp90, gp96, calreticulin, hsp110
or grp170 or noncovalent or covalent complexes thereof complexed to
a peptide.
5.3. Preparation of Heat Shock Proteins and .alpha.2M
[0109] In the present invention, purified unbound HSPs, HSPs
covalently or noncovalently bound to specific peptides or
nonspecific peptides (collectively referred to herein as
HSP-peptide complexes), and combinations of thereof are used.
Purification of HSPs in complexed or non-complexed forms are
described in the following subsections. Further, one skilled in the
art can synthesize HSPs by recombinant expression or peptide
synthesis, which are also described below.
[0110] Also encompassed by the present invention are purified
unbound .alpha.2M, .alpha.2M covalently or noncovalently bound to
specific peptides or nonspecific peptides (collectively referred to
herein as .alpha.2M-peptide complexes), and combinations of thereof
are used. Purification of .alpha.2M in complexed or non-complexed
forms are described in the following subsections. Further, one
skilled in the art can synthesize .alpha.2M by recombinant
expression or peptide synthesis, which are also described
below.
[0111] 5.3.1. Preparation and Purification of Hsp70 or
Hsp70-Peptide Complexes
[0112] The purification of noncovalently bound cellularly produced
hsp70-peptide complexes has been described previously, see, for
example, Udono et al., 1993, J. Exp. Med. 178:1391-1396. A
procedure that may be used, presented by way of example but not
limitation, is as follows:
[0113] Initially, human or mammalian cells are suspended in 3
volumes of 1.times. Lysis buffer consisting of 5 mM sodium
phosphate buffer (pH 7), 150 mM NaCl, 2 mM CaCl.sub.2, 2 mM
MgCl.sub.2 and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Then,
the pellet is sonicated, on ice, until >99% cells are lysed as
determined by microscopic examination. As an alternative to
sonication, the cells may be lysed by mechanical shearing and in
this approach the cells typically are resuspended in 30 mM sodium
bicarbonate (pH 7.5), 1 mM PMSF, incubated on ice for 20 minutes
and then homogenized in a Dounce homogenizer until >95% cells
are lysed.
[0114] Then the lysate is centrifuged at 1,000 g for 10 minutes to
remove unbroken cells, nuclei and other cellular debris. The
resulting supernatant is recentrifuged at 100,000 g for 90 minutes,
the supernatant harvested and then mixed with Con A Sepharose.TM.
equilibrated with phosphate buffered saline (PBS) containing 2 mM
Ca.sup.2+ and 2 mM Mg.sup.2+. When the cells are lysed by
mechanical shearing the supernatant is diluted with an equal volume
of 2.times. lysis buffer prior to mixing with Con A Sepharose.TM..
The supernatant is then allowed to bind to the Con A Sepharose.TM.
for 2-3 hours at 4.degree. C. The material that fails to bind is
harvested and dialyzed for 36 hours (three times, 100 volumes each
time) against 10 mM Tris-Acetate (pH 7.5), 0.1 mM EDTA, 10 mM NaCl,
1 mM PMSF. Then the dialyzate is centrifuged at 17,000 rpm (Sorvall
SS34 rotor) for 20 minutes. Then the resulting supernatant is
harvested and applied to a Mono Q FPLC.TM. ion exchange
chromatographic column (Pharmacia) equilibrated in 20 mM
Tris-Acetate (pH 7.5), 20 mM NaCl, 0.1 mM EDTA and 15 mM
2-mercaptoethanol. The column is then developed with a 20 mM to 500
mM NaCl gradient and then eluted fractions fractionated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
characterized by immunoblotting using an appropriate anti-hsp70
antibody (such as from clone N27F3-4, from StressGen).
[0115] Fractions strongly immunoreactive with the anti-hsp70
antibody are pooled and the hsp70-peptide complexes precipitated
with ammonium sulfate; specifically with a 50%-70% ammonium sulfate
cut. The resulting precipitate is then harvested by centrifugation
at 17,000 rpm (SS34 Sorvall rotor) and washed with 70% ammonium
sulfate. The washed precipitate is then solubilized and any
residual ammonium sulfate removed by gel filtration on a
Sephadex.RTM. G25 column (Pharmacia). If necessary the hsp70
preparation thus obtained can be repurified through the Mono Q
FPLC.TM. ion exchange chromatographic column (Pharmacia) as
described above.
[0116] The hsp70-peptide complex can be purified to apparent
homogeneity using this method. Typically 1 mg of hsp70-peptide
complex can be purified from 1 g of cells/tissue.
[0117] An improved method for purification of hsp70-peptide
complexes comprises contacting cellular proteins with ADP or a
nonhydrolyzable analog of ATP affixed to a solid substrate, such
that hsp70 in the lysate can bind to the ADP or nonhydrolyzable ATP
analog, and eluting the bound hsp70. A preferred method uses column
chromatography with ADP affixed to a solid substratum (e.g.,
ADP-agarose). The resulting hsp70 preparations are higher in purity
and devoid of contaminating peptides. The hsp70 complex yields are
also increased significantly by about more than 10 fold.
Alternatively, chromatography with nonhydrolyzable analogs of ATP,
instead of ADP, can be used for purification of hsp70-peptide
complexes. By way of example but not limitation, purification of
hsp70-peptide complexes by ADP-agarose chromatography can be
carried out as follows:
[0118] Meth A sarcoma cells (500 million cells) are homogenized in
hypotonic buffer and the lysate is centrifuged at 100,000 g for 90
minutes at 4.degree. C. The supernatant is applied to an
ADP-agarose column. The column is washed in buffer and is eluted
with 5 column volumes of 3 mM ADP. The hsp70-peptide complexes
elute in fractions 2 through 10 of the total 15 fractions which
elute. The eluted fractions are analyzed by SDS-PAGE. The
hsp70-peptide complexes can be purified to apparent homogeneity
using this procedure.
[0119] Separation of the HSP from an hsp70-peptide complex can be
performed in the presence of ATP or low pH. These two methods may
be used to elute the peptide from an hsp70-peptide complex. The
first approach involves incubating an hsp70-peptide complex
preparation in the presence of ATP. The other approach involves
incubating an hsp70-peptide complex preparation in a low pH buffer.
These methods and any others known in the art may be applied to
separate the HSP and peptide from an hsp-peptide complex.
[0120] 5.3.2. Preparation and Purification of Hsp90 or Noncovalent
Cellularly Produced Hsp90-Peptide Complexes
[0121] A procedure that can be used, presented by way of example
and not limitation, is as follows:
[0122] Initially, human or mammalian cells are suspended in 3
volumes of 1.times. Lysis buffer consisting of 5 mM sodium
phosphate buffer (pH 7), 150mM NaCl, 2 mM CaCl.sub.2, 2 mM
MgCl.sub.2 and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Then,
the pellet is sonicated, on ice, until >99% cells are lysed as
determined by microscopic examination. As an alternative to
sonication, the cells may be lysed by mechanical shearing and in
this approach the cells typically are resuspended in 30mM sodium
bicarbonate (pH 7.5), 1 mM PMSF, incubated on ice for 20 minutes
and then homogenized in a Dounce homogenizer until >95% cells
are lysed.
[0123] Then the lysate is centrifuged at 1,000 g for 10 minutes to
remove unbroken cells, nuclei and other cellular debris. The
resulting supernatant is recentrifuged at 100,000 g for 90 minutes,
the supernatant harvested and then mixed with Con A Sepharose.TM.
equilibrated with PBS containing 2 mM Ca.sup.2+ and 2 mM Mg.sup.2+.
When the cells are lysed by mechanical shearing the supernatant is
diluted with an equal volume of 2.times. Lysis buffer prior to
mixing with Con A Sepharose.TM.. The supernatant is then allowed to
bind to the Con A Sepharose.TM. for 2-3 hours at 4.degree. C. The
material that fails to bind is harvested and dialyzed for 36 hours
(three times, 100 volumes each time) against 10 mM Tris-Acetate (pH
7.5), 0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF. Then the dialyzate is
centrifuged at 17,000 rpm (Sorvall SS34 rotor) for 20 minutes. Then
the resulting supernatant is harvested and applied to a Mono Q
FPLC.TM. ion exchange chromatographic column (Pharmacia)
equilibrated with lysis buffer. The proteins are then eluted with a
salt gradient of 200 mM to 600 mM NaCl.
[0124] The eluted fractions are fractionated by SDS-PAGE and
fractions containing the hsp90-peptide complexes identified by
immunoblotting using an anti-hsp90 antibody such as 3G3 (Affinity
Bioreagents). Hsp90-peptide complexes can be purified to apparent
homogeneity using this procedure. Typically, 150-200 .mu.g of
hsp90-peptide complex can be purified from 1 g of cells/tissue.
[0125] Separation of the HSP from an hsp90-peptide complex can be
performed in the presence of ATP or low pH. These two methods may
be used to elute the peptide from an hsp90-peptide complex. The
first approach involves incubating an hsp90-peptide complex
preparation in the presence of ATP. The other approach involves
incubating an hsp90-peptide complex preparation in a low pH buffer.
These methods and any others known in the art may be applied to
separate the HSP and peptide from an hsp-peptide complex.
[0126] 5.3.3. Preparation and Purification of Gp96 or Noncovalent
Cellularly Produced Gp96-Peptide Complexes
[0127] A procedure that can be used, presented by way of example
and not limitation, is as follows:
[0128] A pellet of human or mammalian cells is resuspended in 3
volumes of buffer consisting of 30 mM sodium bicarbonate buffer (pH
7.5) and 1 mM PMSF and the cells allowed to swell on ice 20
minutes. The cell pellet is then homogenized in a Dounce
homogenizer (the appropriate clearance of the homogenizer will vary
according to each cell type) on ice until >95% cells are
lysed.
[0129] The lysate is centrifuged at 1,000 g for 10 minutes to
remove unbroken cells, nuclei and other debris. The supernatant
from this centrifugation step is then recentrifuged at 100,000 g
for 90 minutes. The gp96-peptide complex can be purified either
from the 100,000 pellet or from the supernatant.
[0130] When purified from the supernatant, the supernatant is
diluted with equal volume of 2.times. lysis buffer and the
supernatant mixed for 2-3 hours at 4.degree. C. with Con A
Sepharose.TM. equilibrated with PBS containing 2 mM Ca.sup.2+ and 2
mM Mg.sup.2+. Then, the slurry is packed into a column and washed
with 1X lysis buffer until the OD.sub.280 drops to baseline. Then,
the column is washed with 1/3 column bed volume of 10%
.alpha.-methyl mannoside (.alpha.-MM) dissolved in PBS containing 2
mM Ca.sup.2+ and 2 mM Mg.sup.2+, the column sealed with a piece of
parafilm, and incubated at 37.degree. C. for 15 minutes. Then the
column is cooled to room temperature and the parafilm removed from
the bottom of the column. Five column volumes of the .alpha.-MM
buffer are applied to the column and the eluate analyzed by
SDS-PAGE. Typically the resulting material is about 60-95% pure,
however this depends upon the cell type and the tissue-to-lysis
buffer ratio used. Then the sample is applied to a Mono Q FPLC.TM.
ion exchange chromatographic column (Pharmacia) equilibrated with a
buffer containing 5 mM sodium phosphate (pH 7). The proteins are
then eluted from the column with a 0-1M NaCl gradient and the gp96
fraction elutes between 400 mM and 550 mM NaCl.
[0131] The procedure, however, may be modified by two additional
steps, used either alone or in combination, to consistently produce
apparently homogeneous gp96-peptide complexes. One optional step
involves an ammonium sulfate precipitation prior to the Con A
purification step and the other optional step involves
DEAE-Sepharose.TM. purification after the Con A purification step
but before the Mono Q FPLC.TM. step.
[0132] In the first optional step, described by way of example as
follows, the supernatant resulting from the 100,000 g
centrifugation step is brought to a final concentration of 50%
ammonium sulfate by the addition of ammonium sulfate. The ammonium
sulfate is added slowly while gently stirring the solution in a
beaker placed in a tray of ice water. The solution is stirred from
about 1/2 to 12 hours at 4.degree. C. and the resulting solution
centrifuged at 6,000 rpm (Sorvall SS34 rotor). The supernatant
resulting from this step is removed, brought to 70% ammonium
sulfate saturation by the addition of ammonium sulfate solution,
and centrifuged at 6,000 rpm (Sorvall SS34 rotor). The resulting
pellet from this step is harvested and suspended in PBS containing
70% ammonium sulfate in order to rinse the pellet. This mixture is
centrifuged at 6,000 rpm (Sorvall SS34 rotor) and the pellet
dissolved in PBS containing 2 mM Ca.sup.2+ and Mg.sup.2+.
Undissolved material is removed by a brief centrifugation at 15,000
rpm (Sorvall SS34 rotor). Then, the solution is mixed with Con A
Sepharose.TM. and the procedure followed as before.
[0133] In the second optional step, described by way of example as
follows, the gp96 containing fractions eluted from the Con A column
are pooled and the buffer exchanged for 5mM sodium phosphate buffer
(pH 7), 300 mM NaCl by dialysis, or preferably by buffer exchange
on a Sephadex G25 column. After buffer exchange, the solution is
mixed with DEAE-Sepharose.TM. previously equilibrated with 5 mM
sodium phosphate buffer (pH 7), 300 mM NaCl. The protein solution
and the beads are mixed gently for 1 hour and poured into a column.
Then, the column is washed with 5 mM sodium phosphate buffer (pH
7), 300 mM NaCl, until the absorbance at 280 nm drops to baseline.
Then, the bound protein is eluted from the column with five volumes
of 5 mM sodium phosphate buffer (pH 7), 700 mM NaCl. Protein
containing fractions are pooled and diluted with 5 mM sodium
phosphate buffer (pH 7) in order to lower the salt concentration to
175 mM. The resulting material then is applied to the Mono Q
FPLC.TM. ion exchange chromatographic column (Pharmacia)
equilibrated with 5 mM sodium phosphate buffer (pH 7) and the
protein that binds to the Mono Q FPLC.TM. ion exchange
chromatographic column (Pharmacia) is eluted as described
before.
[0134] It is appreciated, however, that one skilled in the art may
assess, by routine experimentation, the benefit of incorporating
the second optional step into the purification protocol. In
addition, it is appreciated also that the benefit of adding each of
the optional steps will depend upon the source of the starting
material.
[0135] When the gp96 fraction is isolated from the 100,000 g
pellet, the pellet is suspended in 5 volumes of PBS containing
either 1% sodium deoxycholate or 1% oxtyl glucopyranoside (but
without the Mg.sup.2+ and Ca.sup.2+) and incubated on ice for 1
hour. The suspension is centrifuged at 20,000 g for 30 minutes and
the resulting supernatant dialyzed against several changes of PBS
(also without the Mg.sup.2+ and Ca.sup.2+) to remove the detergent.
The dialysate is centrifuged at 100,000 g for 90 minutes, the
supernatant harvested, and calcium and magnesium are added to the
supernatant to give final concentrations of 2 mM, respectively.
Then the sample is purified by either the unmodified or the
modified method for isolating gp96-peptide complex from the 100,000
g supernatant, see above.
[0136] The gp96-peptide complexes can be purified to apparent
homogeneity using this procedure. About 10-20 .mu.g of gp96 can be
isolated from 1 g cells/tissue.
[0137] Separation of the HSP from an gp96-peptide complex can be
performed in the presence of ATP or low pH. These two methods may
be used to elute the peptide from an gp96-peptide complex. The
first approach involves incubating an gp96-peptide complex
preparation in the presence of ATP. The other approach involves
incubating an gp96-peptide complex preparation in a low pH buffer.
These methods and any others known in the art may be applied to
separate the HSP and peptide from an hsp-peptide complex.
[0138] 5.3.4. Preparation and Purification of Noncovalent
Cellularly Produced Hsp110-Peptide Complexes
[0139] A procedure, described by Wang et al., 2001, J. Immunol.
166(1):490-7, that can be used, presented by way of example and not
limitation, is as follows:
[0140] A pellet (40-60 ml) of cell or tissue, e.g., tumor cell
tissue, is homogenized in 5 vol of hypotonic buffer (30 mN sodium
bicarbonate, pH7.2, and protease inhibitors) by Dounce
homogenization. The lysate is centrifuged at 4,500.times.g and then
100,000.times.g for 2 hours. If the cells or tissues are of hepatic
origin, the resulting supernatant is was first applied to a blue
Sepharose column (Pharmacia) to remove albumin. Otherwise, the
resulting supernatant is applied to a Con A-Sepharose column
(Pharmacia Biotech, Piscataway, N.J.) previously equilibrated with
binding buffer (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM
MgCl.sub.2; 1 mM CaCl.sub.2; 1 mM MnCl.sub.2; and 15 mM 2-ME). The
bound proteins are eluted with binding buffer containing 15%
.alpha.-D-o-methylmannoside (Sigma, St. Louis, Mo.).
[0141] Con A-Sepharose unbound material is first dialyzed against a
solution of 20 mM Tris-HCl, pH 7.5; 100 mM NaCl; and 15 mM 2-ME,
and then applied to a DEAE-Sepharose column and eluted by salt
gradient from 100 to 500 mM NaCl. Fractions containing hsp110 are
collected, dialyzed, and loaded onto a Mono Q (Pharmacia) 10/10
column equilibrated with 20 mM Tris-HCl, pH 7.5; 200 mM NaCl; and
15 mM 2-ME. The bound proteins are eluted with a 200-500 mM NaCl
gradient. Fractions are analyzed by SDS-PAGE followed by
immunoblotting with an Ab for hsp110, as described by Wang et al.,
1999, J. Immunol. 162:3378. Pooled fractions containing hsp110 are
concentrated by Centriplus (Amicon, Beverly, Mass.) and applied to
a Superose 12 column (Pharmacia). Proteins are eluted by 40 mM
Tris-HCl, pH 8.0; 150 mM NaCl; and 15 mM 2-ME with a flow rate of
0.2 ml/min.
[0142] 5.3.5. Preparation and Purification of Noncovalent
Cellularly Produced Grp170-Peptide Complexes
[0143] A procedure, described by Wang et al., 2001, J. Immunol.
166(1):490-7, that can be used, presented by way of example and not
limitation, is as follows:
[0144] A pellet (40-60 ml) of cell or tissue, e.g., tumor cell
tissue, is homogenized in 5 vol of hypotonic buffer (30 mN sodium
bicarbonate, pH7.2, and protease inhibitors) by Dounce
homogenization. The lysate is centrifuged at 4,500.times.g and then
100,000.times.g for 2 hours. If the cells or tissues are of hepatic
origin, the resulting supernatant is was first applied to a blue
Sepharose column (Pharmacia) to remove albumin. Otherwise, the
resulting supernatant is applied to a Con A-Sepharose column
(Pharmacia Biotech, Piscataway, N.J.) previously equilibrated with
binding buffer (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM
MgCl.sub.2; 1 mM CaCl.sub.2; 1 mM MnCl.sub.2; and 15 mM 2-ME). The
bound proteins are eluted with binding buffer containing 15%
.alpha.-D-o-methylmannoside (Sigma, St. Louis, Mo.).
[0145] Con A-Sepharose-bound material is first dialyzed against 20
mM Tris-HCl, pH 7.5, and 150 mind NaCl and then applied to a Mono Q
column and eluted by a 150 to 400 mM NaCl gradient. Pooled
fractions are concentrated and applied on the Superose 12 column
(Pharmacia). Fractions containing homogeneous grp170 are
collected.
[0146] 5.3.6. .alpha.2M-Antigenic Molecule Complexes
[0147] Endogenous .alpha.2M-antigenic molecule complexes can be
obtained by the following non-limiting methods.
[0148] Alpha-2-macroglobulin can be bought from commercial sources
or prepared by purifying it from human blood. To purify .alpha.2M
from blood, the following non-limiting protocol can be used:
[0149] Blood is collected from a subject and is allowed to clot. It
is then centrifuged for 30 minutes under 14,000.times.g to obtain
the serum which is then applied to a gel filtration column
(Sephacryl S-300R) equilibrated with 0.04M Tris buffer pH 7.6 plus
0.3M NaCl. A 65 ml column is used for about 10 ml of serum. Three
ml fractions are collected and each fraction is tested for the
presence of .alpha.2M by dot blot using an .alpha.2M specific
antibody. The .alpha.2M positive fractions are pooled and applied
to a PD10 column to exchange the buffer to 0.01M Sodium Phosphate
buffer pH 7.5 with PMSF. The pooled fractions are then applied to a
Con A column (10 ml) equilbrated with the phosphate buffer. The
column is washed and the protein is eluted with 5% methylmannose
pyranoside. The eluent is passed over a PD10 column to change the
buffer to a Sodium Acetate buffer (0.05M; pH6.0). A DEAE column is
then equilibrated with acetate buffer and the sample is applied to
the DEAE column. The column is washed and the protein is eluted
with 0.13M sodium acetate. The fractions with .alpha.2M are then
pooled.
[0150] 5.3.6. Recombinant Expression of HSPs and .alpha.2M and
Antigenic Peptides
[0151] Methods known in the art can be utilized to recombinantly
produce HSPs and .alpha.2M. A nucleic acid sequence encoding a heat
shock protein or encoding .alpha.2M can be inserted into an
expression vector for propagation and expression in host cells.
[0152] An expression construct, as used herein, refers to a
nucleotide sequence encoding an HSP or .alpha.2M operably
associated with one or more regulatory regions which enables
expression of the HSP or .alpha.2M in an appropriate host cell.
"Operably-associated" refers to an association in which the
regulatory regions and the HSP or .alpha.2M sequence to be
expressed are joined and positioned in such a way as to permit
transcription, and ultimately, translation.
[0153] The regulatory regions necessary for transcription of the
HSP or .alpha.2M can be provided by the expression vector. A
translation initiation codon (ATG) may also be provided if the HSP
or .alpha.2M gene sequence lacking its cognate initiation codon is
to be expressed. In a compatible host-construct system, cellular
transcriptional factors, such as RNA polymerase, will bind to the
regulatory regions on the expression construct to effect
transcription of the modified HSP or .alpha.2M sequence in the host
organism. The precise nature of the regulatory regions needed for
gene expression may vary from host cell to host cell. Generally, a
promoter is required which is capable of binding RNA polymerase and
promoting the transcription of an operably-associated nucleic acid
sequence. Such regulatory regions may include those 5' non-coding
sequences involved with initiation of transcription and
translation, such as the TATA box, capping sequence, CAAT sequence,
and the like. The non-coding region 3' to the coding sequence may
contain transcriptional termination regulatory sequences, such as
terminators and polyadenylation sites.
[0154] In order to attach DNA sequences with regulatory functions,
such as promoters, to the HSP or .alpha.2M gene sequence or to
insert the HSP or .alpha.2M gene sequence into the cloning site of
a vector, linkers or adapters providing the appropriate compatible
restriction sites may be ligated to the ends of the cDNAs by
techniques well known in the art (Wu et al., 1987, Methods in
Enzymol 152:343-349). Cleavage with a restriction enzyme can be
followed by modification to create blunt ends by digesting back or
filling in single-stranded DNA termini before ligation.
Alternatively, a desired restriction enzyme site can be introduced
into a fragment of DNA by amplification of the DNA by use of PCR
with primers containing the desired restriction enzyme site.
[0155] An expression construct comprising an HSP or .alpha.2M
sequence operably associated with regulatory regions can be
directly introduced into appropriate host cells for expression and
production of HSP-peptide complexes and .alpha.2M-peptide complexes
without further cloning. See, for example, U.S. Pat. No. 5,580,859.
The expression constructs can also contain DNA sequences that
facilitate integration of the HSP or .alpha.2M sequence into the
genome of the host cell, e.g., via homologous recombination. In
this instance, it is not necessary to employ an expression vector
comprising a replication origin suitable for appropriate host cells
in order to propagate and express the HSP or .alpha.2M in the host
cells.
[0156] A variety of expression vectors may be used including, but
not limited to, plasmids, cosmids, phage, phagemids or modified
viruses. Typically, such expression vectors comprise a functional
origin of replication for propagation of the vector in an
appropriate host cell, one or more restriction endonuclease sites
for insertion of the HSP or .alpha.2M gene sequence, and one or
more selection markers. The expression vector must be used with a
compatible host cell which may be derived from a prokaryotic or an
eukaryotic organism including but not limited to bacteria, yeasts,
insects, mammals and humans.
[0157] For long term, high yield production of properly processed
HSP/.alpha.2M or HSP-peptide/.alpha.2M-peptide complexes, stable
expression in mammalian cells is preferred. Cell lines that stably
express HSP/.alpha.2M or HSP-peptide/.alpha.2M-peptide complexes
may be engineered by using a vector that contains a selectable
marker. By way of example but not limitation, following the
introduction of the expression constructs, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
expression construct confers resistance to the selection and
optimally allows cells to stably integrate the expression construct
into their chromosomes and to grow in culture and to be expanded
into cell lines. Such cells can be cultured for a long period of
time while HS/.alpha.2M P is expressed continuously.
[0158] The recombinant cells may be cultured under standard
conditions of temperature, incubation time, optical density and
media composition. However, conditions for growth of recombinant
cells may be different from those for expression of HSPs/.alpha.2M
and antigenic proteins. Modified culture conditions and media may
also be used to enhance production of the HSP/.alpha.2M. For
example, recombinant cells containing HSPs with their cognate
promoters may be exposed to heat or other environmental stress, or
chemical stress. Any techniques known in the art may be applied to
establish the optimal conditions for producing HSP/.alpha.2M or
HSP-peptide/.alpha.2M-peptide complexes.
[0159] Cells may be derived from a variety of sources, including,
but not limited to, cells infected with an infectious agent and
cancer cells and include, but are not limited to, epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
The choice of cell type depends on the type of tumor or infectious
disease being treated or prevented, and can be determined by one of
skill in the art. In a specific embodiment, an expression construct
comprising a nucleic acid sequence encoding the HSP/.alpha.2M
polypeptide is introduced into an antigenic cell. As used herein,
antigenic cells may include cells that are infected with an
infectious agent or pathogen, cells infected with non-infectious or
non-pathogenic forms of an infectious agent or pathogen (e.g., by
use of a helper infectious agent), cells infected by or engineered
to express an attenuated form of an infectious agent or a
non-pathogenic or replication-deficient variant of a pathogen,
pre-neoplastic cells that are infected with a cancer-causing
infectious agent, such as a virus, but which are not yet
neoplastic; or antigenic cells that have been exposed to a mutagen
or cancer-causing agent, such as, for example DNA-damaging agents,
radiation, etc. Other cells that can be used are pre-neoplastic
cells which are in transition from a normal to a neoplastic form as
characterized by morphology, physiological or biochemical
functions. Preferably, the cancer cells and pre-neoplastic cells
used in the methods of the invention are of mammalian origin.
Mammals contemplated by this aspect of the invention include
humans, companion animals (e.g., dogs and cats), livestock animals
(e.g., sheep, cattle, goats, pigs and horses), laboratory animals
(e.g., mice, rats and rabbits), and captive or free wild
animals.
[0160] In various embodiments, any cancer cell, preferably a human
cancer cell, can be used in the present methods for producing the
peptide-complexes. The cancer cells provide the antigenic peptides
which become associated covalently or noncovalently with the
expressed HSP/.alpha.2M polypeptide. The peptide-complexes are then
purified from the cells and used to treat such cancers. Cancers
which can be treated or prevented with immunogenic compositions
prepared by methods of the invention include, but are not limited
to, tumors such as sarcomas and carcinomas. Accordingly, any
tissues or cells isolated from a pre-neoplastic lesion, a cancer,
including cancer that has metastasized to multiple remote sites,
can be used in the present method. For example, cells found in
abnormally growing tissue, circulating leukemic cells, metastatic
lesions as well as solid tumor tissue can be used.
[0161] In another embodiment, cell lines derived from a
pre-neoplastic lesion, cancer tissues or cancer cells can also be
used, provided that the cells of the cell line have at least one or
more antigenic determinants in common with antigens on the target
cancer cells. Cancer tissues, cancer cells, cells infected with a
cancer-causing agent, other pre-neoplastic cells, and cell lines of
human origin are preferred.
[0162] Cancer and pre-neoplastic cells can be identified by any
method known in the art. For example, cancer cells can be
identified by morphology, enzyme assays, proliferation assays,
cytogenetic characterization, DNA mapping, DNA sequencing, the
presence of cancer-causing virus, or a history of exposure to
mutagen or cancer-causing agent, imaging, etc. Cancer cells may
also be obtained by surgery, endoscopy, or other biopsy techniques.
If some distinctive characteristics of the cancer cells are known,
they can also be obtained or purified by any biochemical or
immunological methods known in the art, such as but not limited to
affinity chromatography, and fluorescence activated cell sorting
(e.g., with fluorescently tagged antibody against an antigen
expressed by the cancer cells).
[0163] Cancer tissues, cancer cells or cell lines may be obtained
from a single individual or pooled from several individuals. It is
not essential that clonal, homogeneous, or purified population of
cancer cells be used. It is also not necessary to use cells of the
ultimate target in vivo (e.g., cells from the tumor of the intended
recipient), so long as at least one or more antigenic determinants
on the target cancer cells is present on the cells used for
expression of the HSP/.alpha.2M polypeptide. In addition, cells
derived from distant metastases may be used to prepare an
immunogenic composition against the primary cancer. A mixture of
cells can be used provided that a substantial number of cells in
the mixture are cancer cells and share at least one antigenic
determinant with the target cancer cell. In a specific embodiment,
the cancer cells to be used in expressing an HSP/.alpha.2M
polypeptide are purified.
[0164] 5.3.5. Peptide Synthesis
[0165] An alternative to producing HSP/.alpha.2M by recombinant
techniques is peptide synthesis. For example, an entire
HSP/.alpha.2M, or a peptide corresponding to a portion of an
HSP/.alpha.2M can be synthesized by use of a peptide synthesizer.
Conventional peptide synthesis or other synthetic protocols well
known in the art may be used.
[0166] Peptides having the amino acid sequence of an HSP/.alpha.2M
or a portion thereof may be synthesized by solid-phase peptide
synthesis using procedures similar to those described by
Merrifield, 1963, J. Am. Chem. Soc., 85:2149, During synthesis,
N-.alpha.-protected amino acids having protected side chains are
added stepwise to a growing polypeptide chain linked by its
C-terminal and to an insoluble polymeric support i.e., polystyrene
beads. The peptides are synthesized by linking an amino group of an
N-.alpha.-deprotected amino acid to an .alpha.-carboxyl group of an
N-.alpha.-protected amino acid that has been activated by reacting
it with a reagent such as dicyclohexylcarbodiimide. The attachment
of a free amino group to the activated carboxyl leads to peptide
bond formation. The most commonly used N-.alpha.-protecting groups
include Boc which is acid labile and Fmoc which is base labile.
Details of appropriate chemistries, resins, protecting groups,
protected amino acids and reagents are well known in the art and so
are not discussed in detail herein (See, Atherton, et al., 1989,
Solid Phase Peptide Synthesis: A Practical Approach, IRL. Press,
and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2nd
Ed., Springer-Verlag).
[0167] Purification of the resulting HSP/.alpha.2M is accomplished
using conventional procedures, such as preparative HPLC using gel
permeation, partition and/or ion exchange chromatography. The
choice of appropriate matrices and buffers are well known in the
art and so are not described in detail herein.
5.4. Antigenic Molecules
[0168] The following subsections provide an overview of peptides
that are useful as antigenic/immunogenic components of the
HSP/.alpha.2M-peptide complexes of the invention, and how such
peptides can be identified, e.g., for use in recombinant expression
of the peptides for in vitro complexing of HSPs/.alpha.2M and
antigenic molecules. However, in the practice of the present
invention, the identity of the antigenic molecule(s) of the
HSP/.alpha.2M peptide-complex need not be known, for example when
the HSP/.alpha.2M complex is purified directly from a cancerous
cell or from a tissue infected with a pathogen.
[0169] 5.4.1. Isolation of Antigenic/Immunogenic Components
[0170] It has been found that antigenic peptides and/or components
can be eluted from HSP/.alpha.2M complexes either in the presence
of ATP or low pH. These experimental conditions may be used to
isolate peptides and/or antigenic components from cells which may
contain potentially useful antigenic determinants. Once isolated,
the amino acid sequence of each antigenic peptide may be determined
using conventional amino acid sequencing methodologies. Such
antigenic molecules can then be produced by chemical synthesis or
recombinant methods, purified, and complexed to HSPs in vitro to
form the HSP complexes of the invention.
[0171] Similarly, it has been found that potentially immunogenic
peptides may be eluted from MHC-peptide complexes using techniques
well known in the art (Falk, K. et al., 1990 Nature 348:248-251;
Elliott, T., et al., 1990, Nature 348:195-197; Falk, K., et al.,
1991, Nature 351:290-296).
[0172] Thus, potentially immunogenic or antigenic peptides may be
isolated from either endogenous stress protein-peptide complexes or
endogenous MHC-peptide complexes for use subsequently as antigenic
molecules, by complexing in vitro to HSP/.alpha.2M to form the
HSP/.alpha.2M complexes of the invention. Exemplary protocols for
isolating peptides and/or antigenic components from either of these
complexes are known in the art are described hereinbelow.
[0173] 5.4.2. Peptides From Stress Protein-Peptide Complexes
[0174] Two methods may be used to elute the peptide from a stress
protein-peptide complex.
[0175] One approach involves incubating the stress protein-peptide
complex in the presence of ATP. The other approach involves
incubating the complexes in a low pH buffer.
[0176] Briefly, the complex of interest is centrifuged through a
Centricon 10 assembly (Millipore) to remove any low molecular
weight material loosely associated with the complex. The large
molecular weight fraction may be removed and analyzed by SDS-PAGE
while the low molecular weight may be analyzed by HPLC as described
below. In the ATP incubation protocol, the stress protein-peptide
complex in the large molecular weight fraction is incubated with 10
mM ATP for 30 minutes at room temperature. In the low pH protocol,
acetic acid or trifluoroacetic acid (TFA) is added to the stress
protein-peptide complex to give a final concentration of 10%
(vol/vol) and the mixture incubated at room temperature or in a
boiling water bath or any temperature in between, for 10 minutes
(See, Van Bleek, et al., 1990, Nature 348:213-216; and Li, et al.,
1993, EMBO Journal 12:3143-3151).
[0177] The resulting samples are centrifuged through a Centricon 10
assembly as mentioned previously. The high and low molecular weight
fractions are recovered. The remaining large molecular weight
stress protein-peptide complexes can be reincubated with ATP or low
pH to remove any remaining peptides.
[0178] The resulting lower molecular weight fractions are pooled,
concentrated by evaporation and dissolved in 0.1% TFA. The
dissolved material is then fractionated by reverse phase high
pressure liquid chromatography (HPLC) using for example a VYDAC C18
reverse phase column equilibrated with 0.1% TFA. The bound material
is then eluted at a flow rate of about 0.8 ml/min by developing the
column with a linear gradient of 0 to 80% acetonitrile in 0.1% TFA.
The elution of the peptides can be monitored by OD210 and the
fractions containing the peptides collected.
[0179] 5.4.3. Peptides from MHC-Peptide Complexes
[0180] The isolation of potentially immunogenic peptides from MHC
molecules is well known in the art and so is not described in
detail herein (See, Falk, et al., 1990, Nature 348:248-251;
Rotzsche, et al., 1990, Nature 348:252-254; Elliott, et al., 1990,
Nature 348:191-197; Falk, et al., 1991, Nature 351:290-296; Demotz,
et al., 1989, Nature 343:682-684; Rotzsche, et al., 1990, Science
249:283-287), the disclosures of which are incorporated herein by
reference.
[0181] Briefly, MHC-peptide complexes may be isolated by a
conventional immunoaffinity procedure. The peptides then may be
eluted from the MHC-peptide complex by incubating the complexes in
the presence of about 0.1% TFA in acetonitrile. The eluted peptides
may be fractionated and purified by reverse phase HPLC, as
before.
[0182] The amino acid sequences of the eluted peptides may be
determined either by manual or automated amino acid sequencing
techniques well known in the art. Once the amino acid sequence of a
potentially protective peptide has been determined the peptide may
be synthesized in any desired amount using conventional peptide
synthesis or other protocols well known in the art.
[0183] Peptides having the same amino acid sequence as those
isolated above may be synthesized by solid-phase peptide synthesis
using procedures similar to those described by Merrifield, 1963,
Am. Chem. Soc., 85:2149. During synthesis, N-.alpha.-protected
amino acids having protected side chains are added stepwise to a
growing polypeptide chain linked by its C-terminal and to an
insoluble polymeric support i.e., polystyrene beads. The peptides
are synthesized by linking an amino group of an
N-.alpha.-deprotected amino acid to an .alpha.-carboxy group of an
N-.alpha.-protected amino acid that has been activated by reacting
it with a reagent such as dicyclohexylcarbodiimide. The attachment
of a free amino group to the activated carboxyl leads to peptide
bond formation. The most commonly used N-.alpha.-protecting groups
include Boc which is acid labile and Fmoc which is base labile.
[0184] Briefly, the C-terminal N-.alpha.-protected amino acid is
first attached to the polystyrene beads. The N-.alpha.-protecting
group is then removed. The deprotected .alpha.-amino group is
coupled to the activated .alpha.-carboxylate group of the next
N-.alpha.-protected amino acid. The process is repeated until the
desired peptide is synthesized. The resulting peptides are then
cleaved from the insoluble polymer support and the amino acid side
chains deprotected. Longer peptides can be derived by condensation
of protected peptide fragments. Details of appropriate chemistries,
resins, protecting groups, protected amino acids and reagents are
well known in the art and so are not discussed in detail herein
(See, Atherton, et al., 1989, Solid Phase Peptide Synthesis; A
Practical Approach, IRL Press, and Bodanszky, 1993, Peptide
Chemistry, A Practical Textbook, 2nd Ed., Springer-Verlag).
[0185] Purification of the resulting peptides is accomplished using
conventional procedures, such as preparative HPLC using gel
permeation, partition and/or ion exchange chromatography. The
choice of appropriate matrices and buffers are well known in the
art and so are not described in detail herein.
[0186] 5.4.4. Exogenous Antigenic Molecules
[0187] Molecules that display the antigenicity of a known antigen
of a pathogen or of a tumor-specific or tumor-associated antigen of
a cancer type, e.g. antigens or antigenic portions thereof, can be
selected for use as antigenic molecules, for complexing to
HSP/.alpha.2M, from among those known in the art or determined by
immunoassay to be able to bind to antibody or MHC molecules
(antigenicity) or generate immune response (immunogenicity). To
determine immunogenicity or antigenicity by detecting binding to
antibody, various immunoassays known in the art can be used,
including but not limited to competitive and non-competitive assay
systems using techniques such as radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitin reactions,
immunodiffusion assays, in vivo immunoassays (using colloidal gold,
enzyme or radioisotope labels, for example), western blots,
immunoprecipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays), complement fixation
assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody
binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In a further embodiment, the secondary antibody is
labelled. Many means are known in the art for detecting binding in
an immunoassay and are envisioned for use. In one embodiment for
detecting immunogenicity, T cell-mediated responses can be assayed
by standard methods, e.g., in vitro cytoxicity assays or in vivo
delayed-type hypersensitivity assays.
[0188] Potentially useful antigens or derivatives thereof for use
as antigenic molecules can also be identified by various criteria,
such as the antigen's involvement in neutralization of a pathogen's
infectivity (wherein it is desired to treat or prevent infection by
such a pathogen) (Norrby, 1985, Summary, in Vaccines 85, Lerner, et
al. (eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., pp. 388-389), type or group specificity, recognition by
patients' antisera or immune cells, and/or the demonstration of
protective effects of antisera or immune cells specific for the
antigen. In addition, where it is desired to treat or prevent a
disease caused by pathogen, the antigen's encoded epitope should
preferably display a small or no degree of antigenic variation in
time or amongst different isolates of the same pathogen.
[0189] Preferably, where it is desired to treat or prevent cancer,
known tumor-specific (i.e., expressed in tumor cells) or tumor
associated antigens (i.e., relatively overexpressed in tumor cells)
or fragments or derivatives thereof are used. For example, such
tumor specific or tumor-associated antigens include but are not
limited to KS 1/4 pan-carcinoma antigen (Perez and Walker, 1990, J.
Immunol. 142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415);
ovarian carcinoma antigen (CA125) (Yu, et al, 1991, Cancer Res.
51(2):468-475); prostatic acid phosphate (Miler, et al., 1990,
Nucl. Acids Res. 18(16):4928); prostate specific antigen (Henttu
and Vihko, 1989, Biochem. Biophys. Res. Comm. 160(2):903-910;
Israeli, et al., 1993, Cancer Res. 53:227-230); melanoma-associated
antigen p97 (Estin, et al., 1989, J. Natl. Cancer Inst.
81(6):445-446); melanoma antigen gp75 (Vijayasardahl, et al., 1990,
J. Exp. Med. 171(4):1375-1380); high molecular weight melanoma
antigen (Natali, et al., 1987, Cancer 59:55-63) and prostate
specific membrane antigen. Other exogenous antigens that may be
complexed to HSPs/.alpha.2M include portions or proteins that are
mutated at a high frequency in cancer cells, such as oncogenes
(e.g., ras, in particular mutants of ras with activating mutations,
which only occur in four amino acid residues (12, 13, 59 or 61)
(Gedde-Dahl et al., 1994, Eur. J. Immunol. 24(2):410-414)) and
tumor suppressor genes (e.g., p53, for which a variety of mutant or
polymorphic p53 peptide antigens capable of stimulating a cytotoxic
T cell response have been identified (Gnjatic et al., 1995, Eur. J.
Immunol. 25(6):1638-1642).
[0190] In a specific embodiment, an antigen or fragment or
derivative thereof specific to a certain tumor is selected for
complexing to HSPs/.alpha.2M to form an HSP/.alpha.2M complex for
administration to a patient having that tumor.
[0191] Preferably, where it is desired to treat or prevent viral
diseases, molecules comprising epitopes of known viruses are used.
For example, such antigenic epitopes may be prepared from viruses
including, but not limited to, hepatitis type A, hepatitis type B,
hepatitis type C, influenza, varicella, adenovirus, herpes simplex
type I (HSV-I), herpes simplex type II (HSV-II), rinderpest,
rhinovirus, echovirus, rotavirus, respiratory syncytial virus,
papilloma virus, papova virus, cytomegalovirus, echinovirus,
arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,
rubella virus, polio virus, human immunodeficiency virus type I
(HIV-I), and human immunodeficiency virus type II (HIV-II).
Preferably, where it is desired to treat or prevent bacterial
infections, molecules comprising epitopes of known bacteria are
used. For example, such antigenic epitopes may be prepared from
bacteria including, but not limited to, mycobacteria rickettsia,
mycoplasma, neisseria and legionella.
[0192] Preferably, where it is desired to treat or prevent
protozoal infections, molecules comprising epitopes of known
protozoa are used. For example, such antigenic epitopes may be
prepared from protozoa including, but not limited to, leishmania,
kokzidioa, and trypanosoma.
[0193] Preferably, where it is desired to treat or prevent
parasitic infections, molecules comprising epitopes of known
parasites are used. For example, such antigenic epitopes may be
from parasites including, but not limited to, chlamydia and
rickettsia.
5.5. In Vitro Production of Non-Covalent HSP/.alpha.2M
Complexes
[0194] In an embodiment in which HSPs/.alpha.2M and the peptides
with which they are endogenously associated in vivo are not
employed, complexes of HSPs/.alpha.2M to antigenic molecules are
produced in vitro. As will be appreciated by those skilled in the
art, the peptides either isolated by the aforementioned procedures
or chemically synthesized or recombinantly produced may be
reconstituted with a variety of purified natural or recombinant
stress proteins in vitro to generate immunogenic non-covalent
stress protein-antigenic molecule complexes. Alternatively,
exogenous antigens or antigenic or immunogenic fragments or
derivatives thereof can be complexed to stress proteins. A
preferred, exemplary protocol for complexing a stress protein and
an antigenic molecule in vitro is discussed below.
[0195] In a method which produces non-covalent HSP-antigenic
molecule complexes and .alpha.2M-antigenic molecule complexes, a
complex is prepared according to the method described by Blachere
et al., 1997 J. Exp. Med. 186(8):1315-22, which incorporated by
reference herein in its entirety. Blachere teaches in vitro
complexing of hsps to antigenic molecule. The protocol described in
Blachere can be modified such that the hsp component is substituted
by .alpha.2M. Binder et at. (2001, J. Immunol. 166:4968-72)
demonstrates that the Blachere method yields complexes of .alpha.2M
bound to antigenic molecules.
[0196] Prior to complexing, the HSPs/.alpha.2M are pretreated with
ATP or low pH to remove any peptides that may be associated with
the HSP/.alpha.2M of interest. When the ATP procedure is used,
excess ATP is removed from the preparation by the addition of
apyranase as described by Levy, et al., 1991, Cell 67:265-274, When
the low pH procedure is used, the buffer is readjusted to neutral
pH by the addition of pH modifying reagents.
[0197] The antigenic molecules and the pretreated HSP/.alpha.2M are
admixed to give an approximately 5 antigenic molecule: 1 stress
protein molar ratio. Then, the mixture is incubated for 15 minutes
to 3 hours at 4.degree. to 45.degree. C. in a suitable binding
buffer such as one containing 20 mM sodium phosphate, pH 7.2, 350
mM NaCl, 3 mM MgCl2 and 1 mM phenyl methyl sulfonyl fluoride
(PMSF). The preparations are centrifuged through a Centricon 10
assembly (Millipore) to remove any unbound peptide. The association
of the peptides with the stress proteins can be assayed by
SDS-PAGE, This is the preferred method for in vitro complexing of
peptides isolated from MHC-peptide complexes of peptides
disassociated from endogenous HSP peptide complexes.
[0198] In an alternative embodiment of the invention, preferred for
producing complexes of hsp70 to exogenous antigenic molecules such
as proteins, 5-10 micrograms of purified HSP is incubated with
equimolar quantities of the antigenic molecule in 20 mM sodium
phosphate buffer pH 7.5, 0,5M NaCl, 3 mM MgCl2 and 1 mM ADP in a
volume of 100 microliter at 37.degree. C. for 1 hr. This incubation
mixture is further diluted to 1 ml in phosphate-buffered
saline.
[0199] In an alternative embodiment of the invention, preferred for
producing complexes of gp96 or hsp90 to peptides, 5-10 micrograms
of purified gp96 or hsp90 is incubated with equimolar or excess
quantities of the antigenic peptide in a suitable buffer such as
one containing 20 mM sodium phosphate buffer pH 7.5, 0.5M NaCl, 3
nM MgCl2 at 60-65.degree. C. for 5-20 min. This incubation mixture
is allowed to cool to room temperature and centrifuged one or more
times if necessary, through a Centricon 10 assembly (Millipore) to
remove any unbound peptide.
[0200] Antigenic molecules may be isolated from various sources,
chemically synthesized, or produced recombinantly. Such methods can
be readily adapted for medium or large scale production of the
immunotherapeutic or prophylactic vaccines.
[0201] Following complexing, the immunogenic antigenic molecule
complexes can optionally be assayed in vitro using, for example,
the mixed lymphocyte target cell assay (MLTC) described below. Once
immunogenic complexes have been isolated they can be optionally
characterized further in animal models using the preferred
administration protocols and excipients discussed below.
5.6. Formation of Covalent HSP/.alpha.2M Complexes
[0202] As an alternative to non-covalent complexes of
HSPs/.alpha.2M and antigenic molecules, antigenic molecules may be
covalently attached to HSPs/.alpha.2M. HSP/.alpha.2M peptide
complexes are preferably cross-linked after their purification from
cells or tissues. Covalently linked complexes are the complexes of
choice when a B cell response is desired.
[0203] In one embodiment, HSPs/.alpha.2M are covalently coupled to
antigenic molecules by chemical crosslinking. Chemical crosslinking
methods are well known in the art. For example, in a preferred
embodiment, glutaraldehyde crosslinking may be used. Glutaradehyde
crosslinking has been used for formation of covalent complexes of
peptides and hsps (see Barrios et al., 1992, Eur. J. Immunol. 22:
1365-1372). Preferably, 1-2 mg of HSP peptide complex is
crosslinked in the presence of 0.002% glutaraldehyde for 2 hours.
Glutaraldehyde is removed by dialysis against phosphate buffered
saline (PBS) overnight (Lussow et al., 1991, Eur. J. Immunol. 21:
2297-2302). In one embodiment, the following protocol is used.
Optionally, HSPs may be pretreated with ATP or low pH prior to
complexing, in order to remove any peptides that may be associated
with the HSP polypeptide. Preferably, 1 mg of HSP is crosslinked to
1 mg of peptide in the presence of 0.002% glutaraldehyde for 2
hours. Glutaraldehyde is removed by dialysis against phosphate
buffered saline (PBS) overnight (Lussow et al., 1991, Eur. J.
Immunol. 21: 2297-2302).
[0204] Other methods for chemical crosslinking may also be used, in
addition other methods for covalent attachment of proteins, such as
photocrosslinking (see Current Protocols in Molecular Biology,
Ausubel et al. (eds.), Greene Publishing Associates and Wiley
Interscience, New York).
[0205] In another embodiment, the HSP and specific antigen(s) are
crosslinked by ultraviolet (UV) crosslinking.
[0206] In one embodiment, HSPs are covalently coupled to peptide
fragments by chemical crosslinking. Chemical crosslinking methods
are well known in the art. For example, in a preferred embodiment,
glutaraldehyde crosslinking may be used. Glutaradehyde crosslinking
has been used for formation of covalent complexes of peptides and
HSPs (see Barrios et al., 1992, Eur. J. Immunol. 22: 1365-1372).
Preferably, 1-2 mg of HSP-peptide complex is crosslinked in the
presence of 0.002% glutaraldehyde for 2 hours. Glutaraldehyde is
removed by dialysis against phosphate buffered saline (PBS)
overnight (Lussow et al., 1991, Eur. J. Immunol. 21: 2297-2302).
Alternatively, an HSP and a population of peptides can be
crosslinked by ultraviolet (UV) crosslinking under conditions known
in the art.
[0207] In another embodiment of the invention, a population of
peptides can be complexed to .alpha.2M by incubating the peptide
fragments with .alpha.2M at a 50:1 molar ratio and incubated at
50.degree. C. for 10 minutes followed by a 30 minute incubation at
25.degree. C. Free (uncomplexed) peptides are then removed by size
exclusion filters. Protein-peptide complexes are preferably
measured by a scintillation counter to make sure that on a per
molar basis, each protein is observed to bind equivalent amounts of
peptide (approximately 0.1% of the starting amount of the peptide).
For details, see Binder, 2001, J. Immunol. 166(8):4968-72, which is
incorporated herein by reference in its entirety.
[0208] Alternatively, a population of antigenic peptides can be
complexed to .alpha.2M covalently by methods as described in PCT
publications WO 94/14976 and WO 99/50303 for complexing a peptide
to .alpha.2M, which are incorporated herein by reference in their
entirety. Covalent linking of a population of antigenic peptides to
.alpha.2M can be performed using a bifunctional crosslinking agent.
Such crosslinking agents and methods of their use are also well
known in the art.
[0209] In general, when an .alpha.2M is mixed with a protease,
cleavage of the "bait" region of .alpha.2M takes place, the
proteinase becomes "trapped" by thioesters, and a conformational
change takes place that allows binding of the .alpha.2M complex to
the .alpha.2M receptor. During proteolytic activation of .alpha.2M,
non-proteolytic ligands can become covalently bound to the
activated thioesters. Non-proteolytic ligands can also be
incorporated into the activated .alpha.2M molecule by ammonia or
methylamine during reversal of the nucleophilic activation,
employing heat (Gram and Pizzo, 1998, Biochemistry, 37: 6009-6014).
Such conditions that allow fortuitous trapping of peptides by
.alpha.2M are employed to prepare the .alpha.2M-antigenic complexes
for use in the invention. Methods for such covalent coupling have
been described previously (Osada et al., 1987, Biochem. Biophys.
Res. Commun. 146:26-31; Osada et al., 1988, Biochem. Biophys. Res.
Commun. 150:883; Chu and Pizzo, 1993, J. Immunol. 150:48; Chu et
al., 1994, Ann. N.Y. Acad. Sci. 737:291-307; Mitsuda et al., 1993,
Biochem. Biophys. Res. Commun. 101:1326-1331). Thus in one
embodiment, an .alpha.2M antigenic molecule complex can be prepared
as described by Gron and Pizzo, 1998, Biochemistry, 37: 6009-6014.
The method of Gron and Pizzo yields complexes of .alpha.2M that are
covalently bound to antigenic molecules.
[0210] For example, .alpha.2M polypeptide is mixed with an
antigenic molecule in the presence of a protease, ammonia or other
small amine nucleophiles such as methylamine and ethylamine.
Non-limiting examples of proteases which may be used include
trypsin, porcine pancreatic elastase (PEP), human neutrophil
elastase, cathepsin G, S. aureus V-8 proteinase trypsin,
.alpha.-chymotrypsin, V8 protease, papain, and proteinase K (see
Ausubel et al., eds., in "Current Protocols in Molecular Biology",
Greene Publishing Associates and Wiley Interscience, New York,
17.4.6-17.4.8). A preferred, exemplary protocol for complexing an
.alpha.2M polypeptide and an antigenic molecule in vitro follows.
The antigenic molecules (1 .mu.g-20 mg) and the .alpha.2M
polypeptide (1 .mu.g-20 mg) are mixed together in
phosphate-buffered saline (PBS) (100 .mu.l-5 ml) in the presence of
a protease, such as trypsin (0.92 mg trypsin in approximately 500
.mu.l PBS, to give an approximately 5:1 antigenic
molecule:.alpha.2M polypeptide molar ratio. The mixture is then
incubated for 5-15 minutes at 37.degree. C. 500 .mu.l 4 mg/ml
p-Aphenyl methyl sulfonyl fluoride (p-APMSF) is added to the
solution to inhibit trypsin activity and incubated for 2 hrs at
25.degree. C. The preparations can be centrifuged through a
Centricon 10 assembly (Millipore) to remove any unbound peptide.
Alternatively, free antigenic molecule may be removed by passage
over a gel permeation column. The association of the peptides with
the .alpha.2M polypeptide can be assayed by SDS-PAGE. This is the
preferred method for in vitro complexing of antigenic molecules
isolated from MHC-antigenic molecule complexes, or peptides
disassociated from endogenous .alpha.2M-antigenic molecule
complexes. The foregoing methods could readily be used to generate
HSP-peptide complexes.
5.7. HSP or .alpha.2M Fusion Proteins
[0211] In certain embodiments of the invention, an HSP/.alpha.2M
antigenic molecule complex is a recombinant fusion protein. Such
recombinant fusion proteins, comprised of HSP/.alpha.2 .mu.M
sequences linked to antigenic molecule sequences, may be used in
the methods of the present invention. To produce such a recombinant
fusion protein, an expression vector is constructed using nucleic
acid sequences encoding the HSP/.alpha.2M fused to sequences
encoding an antigenic molecule, using recombinant methods known in
the art (see Suzue et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:
13146-51). HSP/.alpha.2M antigenic peptide fusions are then
expressed and isolated. By specifically designing the antigenic
peptide portion of the molecule, such fusion proteins can be used
to elicit an immune response and in immunotherapy against target
cancer and infectious diseases.
5.8. Kits, Dosage Regimens, Administration and Formulations
[0212] Kits are also provided for carrying out the therapeutic
methods of the present invention. In one embodiment, a kit
comprises a first container containing a purified HSP preparation
or .alpha.2M preparation and a second container containing a
non-vaccine therapeutic modality for treatment of cancer.
Preferably, the cancer is CML, the HSP preparation comprises
hsp70-peptide complexes, and the therapeutic modality is
Gleevec.TM.. In a specific embodiment, the second container
contains imatinib mesylate. In another specific embodiment, the
imatinib mesylate is purified. In a specific embodiment, a kit
comprises a first container containing a purified HSP preparation
or .alpha.2M preparation in an amount ineffective to treat a
disease or disorder when administered alone; and a second container
containing a non-vaccine treatment modality in an amount that, when
administered before, concurrently with, or after the administration
of the HSP preparation or .alpha.2M preparation in the first
container, is effective to improve overall treatment effectiveness
over the effectiveness of the administration of each component
alone. In another specific embodiment, a kit comprises a first
container containing a purified HSP preparation or .alpha.2M
preparation in an amount ineffective to treat a disease or disorder
when administered alone; and a second container containing one or
more non-vaccine treatment modalities in an amount that, when
administered before, concurrently with, or after the administration
of the HSP preparation or .alpha.2M preparation in the first
container, is effective to improve overall treatment effectiveness
over the effectiveness of the administration of the HSP preparation
or .alpha.2M preparation administered alone or the treatment
modalities administered alone. In yet another specific embodiment,
a first container containing a purified HSP preparation or
.alpha.2M preparation in an amount ineffective to treat a disease
or disorder when administered alone; and a second container and
third container, each containing a non-vaccine treatment modality
in an amount that, when administered before, concurrently with, or
after the administration of the HSP preparation or .alpha.2M
preparation in the first container, is effective to improve overall
treatment effectiveness over the effectiveness of the
administration of HSP preparation or .alpha.2M preparation
administered alone or treatment modalities administered alone. In a
preferred specific embodiment, the invention provides a kit
comprising in a first container, a purified HSP preparation or
.alpha.2M comprising a population of noncovalent HSP-peptide
complexes .alpha.2M-peptide complexes obtained from cancerous
tissue of a mammal; in a second container, a composition comprising
a purified cancer chemotherapeutic agent; and in a third container,
a composition comprising a purified cytokine. In a specific
embodiment, the second container containing imatinib mesylate
contains purified imatinib mesylate.
[0213] The dosage of HSP preparation or .mu.2 M preparation to be
administered depends to a large extent on the condition and size of
the subject being treated as well as the amount of non-vaccine
treatment modality administered, the frequency of treatment and the
route of administration. Regimens for continuing therapy, including
site, dose and frequency may be guided by the initial response and
clinical judgment.
[0214] Depending on the route of administration and the type of
HSPs in the HSP preparation, the amount of HSP in the HSP
preparation can range, for example, from 0.1 to 1000 .mu.g per
administration. The preferred amounts of gp96 or hsp70 are in the
range of 10 to 600 .mu.g per administration and 0.1 to 100 .mu.g if
the HSP preparation is administered intradermally. A particularly
preferred amount of hsp70 is about 50 .mu.g per administration if
administered intradermally. For hsp 90, the preferred amounts are
about 50 to 1000 .mu.g per administration, and about 5 to 50 .mu.g
for intradermal administration. The amount of .alpha.2M
administered can range from 2 to 1000 .mu.g, preferably 20 to 500
.mu.g, most preferably about 25 to 250 .mu.g, given once weekly for
about 4-6 weeks, intradermally with the site of administration
varied sequentially.
[0215] Because in certain embodiments, the methods of the invention
use administration of HSP preparation in sub-optimal amounts, it is
envisioned that depending on the route of administration and the
type of HSPs in the HSP preparation, the amount of HSP in the HSP
preparation can be less than an amount in the range of 0.1 to 1000
.mu.g per administration. Accordingly, the preferred amounts of
gp96 or hsp70 are in amounts less than the range of 10 to 600 .mu.g
per administration and less than the range of 0.1 to 10 .mu.g if
the HSP preparation is administered intradermally. For hsp 90, the
preferred amounts are less than the range of 50 to 1000 .mu.g per
administration, and less than the range of 5 to 50 .mu.g for
intradermal administration. The amount of .alpha.2M administered
can range from less than the range of 2 to 1000 .mu.g, preferably
less than the range of 20 to 500 .mu.g, most preferably less than
the range of 25 to 250 .mu.g, given once weekly for about 4-6
weeks, intradermally with the site of administration varied
sequentially.
[0216] Solubility and the site of the administration of the
treatment modality are factors which should be considered when
choosing the route of administration of the HSP preparation of the
invention. The mode of administration can be varied, including, but
not limited to, e.g., subcutaneously, intravenously,
intraperitoneally, intramuscularly, intradermally or mucosally.
Mucosal routes can further take the form of oral, rectal and nasal
administration. With the above factors taken into account, it may
be preferable to administer the HSP to a site that is the same or
proximal to the site of administration of the treatment
modality.
[0217] In an embodiment of the invention, HSPs/.alpha.2M may be
administered using any desired route of administration. Advantages
of intradermal administration include use of lower doses and rapid
absorption, respectively. Advantages of subcutaneous or
intramuscular administration include suitability for some insoluble
suspensions and oily suspensions, respectively. Mucosal routes of
administration include, but are not limited to, oral, rectal and
nasal administration. Preparations for mucosal administrations are
suitable in various formulations as described below.
[0218] If the HSP/.alpha.2M preparation is water-soluble, then it
may be formulated in an appropriate buffer, for example, phosphate
buffered saline or other physiologically compatible solutions,
preferably sterile. Alternatively, if the resulting complex has
poor solubility in aqueous solvents, then it may be formulated with
a non-ionic surfactant such as Tween, or polyethylene glycol. Thus,
the compounds and their physiologically acceptable solvates may be
formulated for administration by inhalation or insufflation (either
through the mouth or the nose) or oral, buccal, parenteral, or
rectal administration or, in the case of tumors, directly injected
into a solid tumor.
[0219] For oral administration, the pharmaceutical preparation may
be in liquid form, for example, solutions, syrups or suspensions,
or may be presented as a drug product for reconstitution with water
or other suitable vehicle before use. Such a liquid preparation may
be prepared by conventional means with pharmaceutically acceptable
additives such as suspending agents (e.g., sorbitol syrup,
cellulose derivatives or hydrogenated edible fats); emulsifying
agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g.,
almond oil, oily esters, or fractionated vegetable oils); and
preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic
acid). The pharmaceutical preparation may take the form of, for
example, tablets or capsules prepared by conventional means with
pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinized maize starch, polyvinyl pyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art.
[0220] The HSP/.alpha.2M preparation for oral administration may be
suitably formulated to give controlled release of the active
compound.
[0221] For buccal administration, the preparation may take the form
of tablets or lozenges formulated in conventional manner.
[0222] The preparation may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The preparation may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0223] The preparation may also be formulated in a rectal
preparation such as a suppository or retention enema, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0224] In addition to the formulations described previously, the
preparation may also be formulated as a depot preparation. Such
long acting formulations may be administered by implantation (for
example, subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the preparation may be formulated
with suitable polymeric or hydrophobic materials (for example, as
an emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt. Liposomes and emulsions are well known examples of delivery
vehicles or carriers for hydrophilic drugs.
[0225] For administration by inhalation, the preparation for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0226] The preparation may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the HSP preparation or .alpha.2M preparation. The pack
may for example comprise metal or plastic foil, such as a blister
pack. The pack or dispenser device may be accompanied by
instructions for administration.
[0227] The appropriate and recommended dosages, formulation and
routes of administration for treatment modalities such as
chemotherapeutic agents, radiation therapy and
biological/immunotherapeutic agents such as cytokines are known in
the art and described in such literature as the Physician's Desk
Reference (56.sup.th ed., 2002). In particular embodiments, the
present invention comprises administering an anti-cancer agent such
as any of those described below in Table 2, preferably for the
treatment of breast, ovary, melanoma, prostate, colon or lung
cancer, CML or soft tissue sarcomas, including but not limited to
gastrointestinal stromal tumors as described below in section
5.11.
[0228] Because in certain embodiments, the methods of the invention
comprise administration of sub-optimal amounts of the therapeutic
modality, it is envisioned that the dosages of each therapeutic
modality can be less than that used in standard therapy or known in
the art.
[0229] In one embodiment, Gleevec.TM. is administered 50 mg to 100
mg, 100 mg to 200 mg, 200 mg to 300 mg, 300 mg to 400 mg, 400 mg to
500 mg, 500 mg to 600 mg, 600 mg to 700 mg, 700 mg to 800 mg, 800
mg to 900 mg, or 900 mg to 1000 mg daily. In certain embodiments,
the total daily dose is administered to a subject as two daily
doses of 25 mg to 50 mg, 50 mg to 100 mg, 100 mg to 200 mg, 200 mg
to 300 mg, 300 mg to 400 mg, or 400 mg to 500 mg. Gleevec.TM. is
administered orally in dosages of 100 mg to 1000 mg, preferably 200
mg to 900 mg, more preferably 300 mg to 800 mg, most preferably 400
mg to 600 mg. In a specific embodiment, Gleevec.TM. is administered
orally, at a sub-optimal daily dosage. In preferred embodiments,
the sub-optimal daily dosage of orally administered Gleevec.TM. is
about 10 mg to 600 mg, about 50 mg to 400 mg, about 100 mg to 300
mg, or about 200 mg. In other embodiments, Gleevec.TM. is
administered orally every other day, every third day, every fourth
day, every fifth day, every sixth day, or once a week, at a dosage
of 100 mg to 800 mg, 200 mg to 600 mg, 300 mg to 500 mg, or 400
mg.
TABLE-US-00002 TABLE 2 Therapeutic Agent
Dose/Administration/Formulation imatinib mesylate Oral 400-600 mg
daily (Gleevec .TM.) (capsule) Capsules each contain imatinib
mesylate equivalent to 100 mg imatinib free base doxorubicin
Intravenous 60-75 mg/m.sup.2 on Day 1 21 day intervals
hydrochloride (Adriamycin RDF .RTM. and Adriamycin PFS .RTM.)
epirubicin Intravenous 100-120 mg/m.sup.2 on Day 1 of each 3-4 week
cycles hydrochloride cycle or (Ellence .TM.) divided equally and
given on Days 1-8 of the cycle fluorousacil Intravenous How
supplied: 5 mL and 10 mL vials (containing 250 and 500 mg
flourouracil respectively) docetaxel Intravenous 60-100 mg/m.sup.2
over 1 hour Once every 3 weeks (Taxotere .RTM.) paclitaxel
Intravenous 175 mg/m.sup.2 over 3 hours Every 3 weeks for (Taxol
.RTM.) 4 courses (administered sequentially to doxorubicin-
containing combination chemotherapy) tamoxifen citrate Oral 20-40
mg Daily (Nolvadex .RTM.) (tablet) Dosages greater than 20 mg
should be given in divided doses (morning and evening) leucovorin
calcium Intravenous or How supplied: Dosage is unclear from text.
for injection intramuscular 350 mg vial PDR 3610 injection
luprolide acetate Single 1 mg (0.2 mL or 20 unit mark) Once a day
(Lupron .RTM.) subcutaneous injection flutamide Oral (capsule) 250
mg 3 times a day at 8 hour (Eulexin .RTM.) (capsules contain 125 mg
intervals (total daily dosage flutamide each) 750 mg) nilutamide
Oral 300 mg or 150 mg 300 mg once a day for 30 (Nilandron .RTM.)
(tablet) (tablets each contain 50 or 150 days followed by 150 mg mg
nilutamide) once a day bicalutamide Oral 50 mg Once a day (Casodex
.RTM.) (tablet) (tablets each contain 50 mg bicalutamide)
progesterone Injection USP in sesame oil 50 mg/mL ketoconazole
Cream 2% cream applied once or twice (Nizoral .RTM.) daily
depending on symptoms prednisone Oral Initial dosage may vary from
5 (tablet) mg to 60 mg per day depending on the specific disease
entity being treated estramustine Oral 14 mg/kg of body weight
(i.e. Daily given in 3 or 4 divided phosphate sodium (capsule) one
140 mg capsule for each 10 doses (Emcyt .RTM.) kg or 22 lb of body
weight) etoposide or Intravenous 5 mL of 20 mg/mL solution VP-16
(100 mg) dacarbazine Intravenous 2-4.5 mg/kg Once a day for 10
days. (DTIC-Dome .RTM.) May be repeated at 4 week intervals
polifeprosan 20 wafer placed 8 wafers, each containing 7.7 mg with
carmustine in resection of carmustine, for a total of 61.6 implant
(BCNU) cavity mg, if size and shape of (nitrosourea) resection
cavity allows (Gliadel .RTM.) cisplatin Injection [n/a in PDR 861]
How supplied: solution of 1 mg/mL in multi- dose vials of 50 mL and
100 mL mitomycin Injection supplied in 5 mg and 20 mg vials
(containing 5 mg and 20 mg mitomycin) gemcitabine HCl Intravenous
For NSCLC-2 schedules have 4 week schedule- (Gemzar .RTM.) been
investigated and the Days 1, 8 and 15 of each 28- optimum schedule
has not been day cycle. Cisplatin determined intravenously at 100
mg/m.sup.2 4 week schedule- on day 1 after the infusion of
administration intravenously at Gemzar. 1000 mg/m.sup.2 over 30
minutes on 3 week schedule- 3 week schedule- Days 1 and 8 of each
21 day Gemzar administered cycle. Cisplatin at dosage of
intravenously at 1250 mg/m.sup.2 100 mg/m.sup.2 administered over
30 minutes intravenously after administration of Gemzar on day 1.
carboplatin Intravenous Single agent therapy: Every 4 weeks
(Paraplatin .RTM.) 360 mg/m.sup.2 I.V. on day 1 (infusion lasting
15 minutes or longer) Other dosage calculations: Combination
therapy with cyclophosphamide, Dose adjustment recommendations,
Formula dosing, etc. ifosamide Intravenous 1.2 g/m.sup.2 daily 5
consecutive days (Ifex .RTM.) Repeat every 3 weeks or after
recovery from hematologic toxicity topotecan Intravenous 1.5
mg/m.sup.2 by intravenous 5 consecutive days, starting
hydrochloride infusion over 30 minutes daily on day 1 of 21 day
course (Hycamtin .RTM.)
5.10. Treatment and Prevention of Infectious Diseases
[0230] Infectious diseases that can be treated using the methods of
the present invention are caused by infectious agents including,
but not limited to, viruses, bacteria, fungi protozoa and
parasites.
[0231] Infectious agents that can be treated according to the
invention include, but are not limited to viruses, bacteria, fungi,
and agents of protozoal disease.
[0232] Viral diseases that can be treated or prevented using the
methods of the present invention include, but are not limited to,
those caused by hepatitis type A, hepatitis type B, hepatitis type
C, influenza, varicella, adenovirus, herpes simplex type I (HSV-1),
herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus,
rotavirus, respiratory syncytial virus, papilloma virus, papova
virus, cytomegalovirus, echinovirus, arbovirus, huntavirus,
coxsackie virus, mumps virus, measles virus, rubella virus, polio
virus, small pox, Epstein Barr virus, human immunodeficiency virus
type I (HIV-I), human immunodeficiency virus type II (HIV-II), and
agents of viral diseases such as viral meningitis, encephalitis,
dengue or small pox.
[0233] Bacterial diseases that can be treated or prevented by use
of the methods of the present invention are caused by bacteria
including, but not limited to, mycobacteria rickettsia, mycoplasma,
neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease),
Bacillus antracis (anthrax), tetanus, streptococcus,
staphylococcus, mycobacterium, tetanus, pertissus, cholera, plague,
diptheria, chlamydia, S. aureus and legionella.
[0234] Protozoal diseases that can be treated or prevented by use
of an immunoreactive reagent in conjunction with the methods of the
present invention are caused by protozoa including, but not limited
to, leishmania, kokzidioa, trypanosoma or malaria.
[0235] Parasitic diseases that can be treated or prevented by use
of the methods of the present invention are caused by parasites
including, but not limited to, chlamydia and rickettsia.
5.11. Treatment of Cancer
[0236] A number of non-vaccine cancer treatment modalities are
currently in clinical trials and well-known in the art. The
HSP/.alpha.2M preparation can be used in conjunction with such
non-vaccine cancer treatment modalities for the treatment and
prevention of the respective types of cancers. One skilled in the
art would be able to determine experimental and standard
anti-cancer therapies and treatments that could be used according
to the methods of the present invention.
[0237] Cancers that can be treated using the methods of the present
invention include, but are not limited to human sarcomas and
carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, testicular tumor, lung carcinoma, small
cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
retinoblastoma, leukemias, acute lymphocytic leukemia, acute
myelocytic leukemia, myeloblastic leukemia, promyelocytic leukemia,
myelomonocytic leukemia, monocytic leukemia, erythroleukemia,
chronic leukemia, chronic myeloid leukemia, chronic myelogenous
leukemia, chronic myelocytic leukemia, chronic granulocytic
leukemia, chronic lymphocytic leukemia, polycythemia vera,
lymphoma, Hodgkin's disease lymphoma, non-Hodgkin's disease
lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy
chain disease, soft tissue sarcomas, gastrointestinal stromal
tumors, and glioblastomas.
6. EXAMPLE
TUMORS NON-RESPONSIVE TO CHEMOTHERAPY/CYTOKINE TREATMENT RESPOND
AFTER ADMINISTRATION OF HSP-PEPTIDE COMPLEX
[0238] Mice bearing tumors, such as LLC (D122) and B16, do not
respond to treatment of Cyclophosphamide (Cy) in combination with
interleukin-12 (IL-12). In a double-graft experiment, mice were
injected with MCA207 (tumors known to respond to Cy+IL-12
treatment) and D122 at two opposite flanks, and tumors were allowed
to grow to a significant size (10.times.10 mm), then the mice were
treated with Cy+IL-12. The large MCA207 tumors regressed rapidly,
whereas the D122 tumors continued to grow on the opposite flank of
the same animals. The results demonstrated that certain tumors,
e.g., D122 do not respond to Cy+IL-12 treatment, even though a
vigorous response against another tumor is present in the same
animal.
[0239] The tumors that responded to the treatment appear to be
immunogenic, whereas other tumors that are non-responders were all
poorly immunogenic. To test whether a host-derived immune
recognition of the tumor in the forms of T cell priming prior to
Cy+IL-12 treatment would result in the tumor responding to
treatment, the following experiments were conducted. The following
results demonstrated that if a mouse bearing a D 122 tumor that
does not respond to Cy+IL-12 treatment alone obtains immunological
memory of the tumor, a tumor rejection will occur in the mice
following treatment with Cy+IL-12.
[0240] Heat shock protein-peptide complexes were used for eliciting
a robust T cell response that includes both CD4+ and CD8+ T cells
in mice.
6.1 Materials and Methods
[0241] Naive mice were either un-immunized, or immunized once at
day 0 with 5 and 20 .mu.g D 122-derived gp96-peptide complexes
administered subcutaneously, or 2 .mu.g D122-derived gp96-peptide
complexes administered intradermally. As a negative control,
another group of mice were immunized with liver-derived
gp96-peptide complexes. The D122-derived gp96-peptide complexes are
HSP-peptide complexes endogenous to and isolated from D122 tumor
cells. The liver-derived gp96-peptide complexes are HSP-peptide
complexes endogenous to and isolated form liver cells. Two weeks
after the immunization (day 14), the mice were challenged
subcutaneously with 200,000 D122 cells. The immunization was
sub-optimal for tumor rejection according to our previous
experience and D122 tumors grew in all mice. When tumor size
reached 10 mm or above in diameter (day 32-34), the mice were
treated with Cy+IL-12 (Cy, 3 mg by intraparenteral administration;
IL-12, 200 ng, intraparenteral administration for 5 days).
6.2. Results
TABLE-US-00003 [0242] Cure rate Size of tumors cured Mice immunized
with (#/total) (mm in diameter) PBS 2/16 7 and 10 Liver-derived
gp96-peptide complexes 2/10 10 and 12 D122-derived gp96-peptide
complexes 11/12 From 8 to 22
[0243] As summarized in the table above, upon antigen-specific
immunological stimulation with autologous tumor derived
gp96-peptide complexes, non-responder tumor D122 became a responder
to the treatment of Cy+IL-12. In groups un-immunized and immunized
with liver-derived gp96-peptide complexes, only those mice bearing
the smallest tumors (less than 10-12 mm in diameter) experienced
tumor regression after Cy+IL-12 treatment. In contrast, in mice
that were immunized with D 122-derived gp96-peptide complexes,
large D122 tumors such as those 22 mm in diameter, which are
generally refractory to any kind of immunotherapeutic approaches
reported, regressed completely after the Cy+IL-12 treatment. In
addition, immunohistochemistry analysis for a number of tumor
samples harvested from each group reveals that 1) No sign of T cell
infiltration in the tumors removed from mice that were un-immunized
or immunized with liver-derived gp96-peptide complexes before or
after the Cy+IL-12 treatment; 2) In contrast, some T cell
infiltration (both CD4+ and CD8+) was observed in tumors harvested
from mice immunized with D122-derived gp96-peptide complexes 12
days, but not 6 days, after the Cy+IL-12 treatment was
initiated.
7. EXAMPLE
COMPLETE ELIMINATION OF LEUKEMIA CELLS IN PATIENTS IN CHRONIC PHASE
CML AFTER ADMINISTRATION OF COMBINATION GLEEVEC.TM. AND HSP-PEPTIDE
COMPLEX
[0244] To test the feasibility of immunization with autologous
tumor-derived hsp70-peptide complexes to treat patients in chronic
phase CML, the following protocol was used (FIG. 1). The clinical
protocol summarized in FIG. 1 includes all physical examinations,
blood work, x-rays and bone marrows that were done before, during
and after vaccination with an HSP preparation. Prior to inclusion
in the study, subjects' diagnosis of CML was confirmed by bcr/abl
molecular typing of peripheral blood or bone marrow obtained from
the subject using polymerase chain reaction (PCR) to determine the
presence or absence of bcr/abl chimeric proteins or
transcripts.
7.2 Materials and Methods
[0245] Subjects that participated fulfilled the following criteria:
subject displayed an Eastern Cooperative Oncology Group (ECOG)
performance score less than 2; subject was at least 18 years of
age, and capable of giving informed consent; less than one year has
passed since the original diagnosis of Philadelphia chromosome
positive CML in chronic phase; subject was not in cytogenetic
remission; subject was not anticipating a bone marrow or stem cell
transplant within the next six months unless such therapy was
deemed necessary by a treatment physician due to evolution of the
disease; subjects were allowed to maintain concurrent standard
treatment hydroxyurea, Ara-C/day for 10 days or Gleevec.TM.
(imatinib mesylate); subject lacked any serious illness such that
medical condition might be compromised by participation in the
study; subject showed adequate renal function as measured by serum
creatinine levels less than 2.0, and adequate hepatic function, as
measured by bilirubin and transaminase less than 2.0 times the
upper normal limit; subject was not on corticosteroid therapy, or
other immunosuppressive medication; and subject did not display a
lack of energy as shown by adequate delayed type hypersensitivity
(DHT) response to at least 1 out of 3 antigens by skin testing with
Candida, mumps and PPD, i.e., induration was greater than 0.5 cm 48
hours after placement.
[0246] Subjects were excluded if: subject displayed an ECOG
performance score.gtoreq.2; subject was more than 3 years out from
original diagnosis of Philadelphia chromosome positive CML in
chronic phase; subject was on IFN treatment; subject showed
significant anemia, i.e., hemoglobin less than 10 g/dl or
thrombocytopenia, i.e., platelet less than 20,000/.mu.l, requiring
transfusion; subject showed peripheral blast count over 10%;
subject showed positive urine or blood pregnancy test; subject
showed impaired renal function, i.e., serum creatine greater than
or equal to 2.0, or impaired hepatic function, i.e., bilirubin or
transaminase more than 2.0 times the upper normal limit; subject
showed significant active infection requiring hospitalization at
time of enrollment; subject with significant behavioral or
psychological problems that prevented adequate follow-up.
[0247] A subject was discontinued for any of the following reasons:
subject requested to withdraw for any reason; a proven effective
therapeutic approach became available, and was preferred by the
subject (e.g., the approval of other investigational medications by
the regulatory agency, identification of an identical human
leukocyte antigen (HLA) matched donor), the subject was lost to
follow-up; the subject showed clear evidence of disease
acceleration despite concurrent therapy as evidenced by the
following signs and symptoms: peripheral blasts 10% or more;
peripheral blast plus promyelocytes 30% or more; peripheral
basophils 20% or more; thrombocytopenia less than 100,000/mm.sup.3
unrelated to therapy; neutropenia less than 1,000/mm.sup.3
unrelated to therapy; marrow blast 10% or more; significant marrow
fibrosis; progressive splenomagly unresponsive to therapy; triad of
WBC greater than 50,000/mm.sup.3, hematocrit less than 25% and
platelets less than 100,000/mm.sup.3 not controlled with therapy;
persistent unexplained fever; and cytogenetic clonal evolution;
extramedullary disease with localized immature blast such as
chloroma; any other reason which in the opinion of the investigator
was to protect the best interest of the subject.
[0248] Prior to administration of their first HSP preparation,
subjects had been receiving Gleevec.TM. therapy (400-800 mg daily
in capsule form, 400-600 mg daily doses administered once a day, or
800 mg in two daily doses of 400 mg each) for 2 days, 5 months, 9
months, 10 months, and 1 year, respectively. Subjects satisfying
the above criteria were allowed to remain on Gleevec.TM. therapy
throughout the study. Subjects subsequently underwent aphaeresis
using peripheral vein access to collect peripheral mononuclear
cells. The majority of the specimen was used for the purification
of Hsp70-peptide complexes. The autologous hsp70-peptide complexes
were then purified using an ADP-agarose protocol, substantially as
described in Section 5.3.1 above. A small fraction of the
collection was used as targets for a CTL, assay. Subjects received
an intradermal injection of 50 .mu.g hsp70-peptide complexes in the
skin of the forearm weekly over a two month period for a total of 8
injections, in addition to Gleevec.TM. therapy (400-800 mg daily in
capsule form, 400-600 mg daily doses administered once a day, 800
mg doses administered twice a day). Blood samples were drawn three
times to access the status of the immune system. Blood was
collected prior to the vaccination, during the vaccination and 1-2
weeks after the 8.sup.th vaccination (see FIG. 1). At the end of
the treatment, all subjects underwent full hematological and
cytogenetic staging on the bone marrow (see Silver et al., 1999,
Blood 94(5):1517-1536).
[0249] In addition, to collect feasibility and toxicity data, the
development of anti-tumor immunity was measured according to
methods known in the art, such as: (1) an increase in peripheral
blood of IFN-.gamma. producing CD8+ T-lymphocytes which are
reactive to the autologous bcr/abl positive peripheral mononuclear
cells (see e.g., Janetzki et al., 2000, Int. J. Cancer 88:232-238);
(2) an increase of PR-1 specific CTLs by PR1-HLA-A2 tetramer
techniques in patients who are HLA-A2 positive (see e.g., Clark et
al., 2001, Blood 98(10): 2887-2893 and Molldrem et al., 1999,
Cancer Research 59: 2675-2681); (3) the change of immunophenotype
of peripheral lymphocytes (see e.g., Akel et al., 2002, Clin. Lab.
Haem. 24:362-367); and (4) the cytogenetic remission of
Philadelphia chromosome from the bone marrow (see e.g., Wang at,
2002, British J. Haematology 118:771-777).
[0250] Combined treatment in the five evaluable subjects resulted
in complete elimination of leukemia cells as determined by: RT-PCR
analysis determining the presence or absence of bcr/abl transcripts
in the peripheral blood or bone marrow collected from treated
patients, (see e.g., Merx et al., 2002, Leukemia 16:1579-1583; Wang
et al., 2002, British Journal of Haematology 118: 771-777; and
Stentoft et al, 2001, Eur. J. Haemotol. 67: 302-308); cytogenetic
response, one of the criteria used in the approval of Gleevec.TM.
(see Silver et al, 1999, Blood 94(5):1517-1536); or a combination
of RT-PCR and cytogenetic response. Based on previous reports, less
than 10 percent of patients treated with Gleevec.TM. alone achieve
responses using these same criteria. See Druker et al., 2002,
Hematology (Am. Soc. Hematol. Educ. Program): 111-135, at
114-115.
[0251] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication or patent or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
[0252] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended claims
along with the full scope of equivalents to which such claims are
entitled.
* * * * *